Team:Duke

From 2009.igem.org

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=Protocols=
=Protocols=
-
==<b>PCR : Phusion Polymerase</b>==
+
==<b>CPEC Cloning</b>==
[[#Protocols | Back to top]]
[[#Protocols | Back to top]]
-
PCR<br>
+
CPEC cloning
-
Phusion Polymerase<br>
+
Materials:
-
Reagent                          Amount<br>
+
• Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
-
ddH20                            35ul<br>
+
• Thermocycler
-
5xHF buffer                      10ul<br>
+
 
-
10mM dntps                        1ul<br>
+
Protocol:
-
20uM Oligo F                      1.25ul<br>
+
5x Phusion HF Buffer 4 ul
-
20uM Oligo R                      1.25ul<br>
+
10 mM dNTPs             0.4 ul
-
Template (1:20 dilution from PCR) 1ul<br>
+
Vector 50 ng/1kb
-
phusion polymerase                0.5ul<br>
+
Insert x ng*
 +
Phusion DNA Polymerase 0.2 ul
 +
H2O           to 20 ul
 +
 
 +
*The amount of insert is determined so that the molar ratio for vector and insert is 1 to 2.
 +
 
 +
98°C    30sec
 +
10X
 +
98°C                        10 sec
 +
Annealing**          30 sec
 +
72°C                        x sec***
 +
72°C    5min
 +
4°C    hold
 +
** Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.
 +
***The extension time is usually calculated according to the shortest piece with 15 sec /kb if the cloning is not complicated. For example, if there is only one insert and is shorter than the vector, say, 600 bp, then I will use 15 sec for extension. Refer to the published paper for detailed information.
-
Initial Denaturation  98C 30sec 1 cycles<br>
 
-
Denaturation 98C 5-10s<br>
 
-
Annealing 45-72C* 10-30s  25-35 cycles<br>
 
-
Extension 72C 15-30s/kb<br>
 
-
Final extension 72C/4C        5-10min  1<br>
 
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
-
==<b>Miniprep</b>==
+
==<b>DNA Purification</b>==
 +
[[#Protocols | Back to top]]<br>
<br>
<br>
-
Preliminary Steps
 
-
  1.
+
DNA purification
-
      Prepare 5 ml LB in a culture tube.
+
Materials :
-
  2.
+
• E.Z.N.A Gel Purification Kit (Omega Bio-Tek, Cat No. D2500-02 )
-
      Pick out and add a single colony with ~20μl pipette.
+
• Water bath equilibrated to 55-65C
-
  3.
+
• Microcentrifuge capable of at least 10,000 x g
-
      Place in shaker for overnight growth. (Plasmid culture should be a typical density of A600=2.0 or higher by then)
+
• Nuclease-free 1.5 ml centrifuge bottles
 +
• Absolute (95%-100%) ethanol
 +
• Protective eye-wear
 +
• Isopropanol (for fragments < 500 bp only)
 +
Protocol:
 +
1. Perform agarose gel electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended, however, that fresh TAE buffer or TBE buffer be used as running buffer. Do not re-use running buffer as its pH will increase and reduce yields.
 +
2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean scalpel.
 +
3. Determine the approximate volume of the gel slice by weighing it in a clean 1.5 ml microfuge tube. Assuming a density of 1 g/ml of gel, the volume of gel is derived as follows: A gel slice of mass 0.3 g will have a volume of 0.3 ml. Add equal volume of Binding Buffer (XP2). Incubate the mixture at 55C-60C for 7 min or until the gel has completely melted. Mix by shaking or inverting the tube every 2-3 minutes. Centrifuge the tube briefly to collect all the liquid to the bottom of the tube.
 +
Note: For DNA fragment less than 500bp, add 1 sample volume of isopropanol after the addition of Binding Buffer (XP2).
 +
4. Apply up to 700 ul of the DNA/agarose solution to a HiBind® DNA spin column assembled in a clean 2 ml collection tube (provided) and centrifuge in a microcentrifuge at 8,000-10,000 x g for 1 min at room temperature. Discard the liquid. Re-use the collection tube in Steps 5-8. For volumes greater than 700 ul, load the column and centrifuge successively, 700 ul at a time. Each HiBind® spin-column has a total capacity of 25-30 ug DNA.
 +
5. Discard liquid and add 300ul Binding Buffer. Centrifuge at 10,000 x g for 1 minutes.
 +
6. Add 700 ul of SPW Buffer diluted with absolute ethanol into the column and wait 2-3 minutes. Centrifuge at 10,000 x g for 1 min at room temperature to wash the sample.
 +
7. Discard liquid and repeat step 6 with another 700 ul SPW Buffer.
 +
8. Discard liquid and, re-using the collection tube, centrifuge the empty column for 1 min at maxi speed (>13,000 x g)  to dry the column matrix. This drying step is critical for good DNA yields.
 +
9. Place column into a clean 1.5 ml microcentrifuge tube (not provided). Add 30-50 ul depending on desired concentration of final product) Elution Buffer (or sterile deionized water) directly to the center of the column matrix, then incubate for 1 minute. Centrifuge 1 min at maxi speed (>13,000 x g) to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lo
-
The Procedure
 
-
  1.
+
==<b>PCA (Polymerase Cycle Assembly</b>==
-
      Label 2 2ml microcentrifuge tubes on the caps with plasmid names.
+
[[#Protocols | Back to top]]<br>
-
  2.
+
-
      Add 1.5 ml harvested culture to each of 2 tubes.
+
-
  3.
+
-
      Insert in microcentrifuge so that they are balanced, and the hinges face away from the center.
+
-
  4.
+
-
      Microcentrifuge at 15,000 rpm for 1 minute (Our centrifuge is limited to 13,000 rpm, but that is fine).
+
-
  5.
+
-
      Pour off supernatant.
+
-
  6.
+
-
      Add another ~.75 ml culture to the 2 tubes.
+
-
  7.
+
-
      Re-centrifuge (at same settings and positions)
+
-
  8.
+
-
      Pour off supernatant.
+
-
  9.
+
-
      Re-centrifuge
+
-
  10.
+
-
      Use a small volume, bore tip pipette for carefully removing supernatant.
+
-
          *
+
-
            The cells are pelleted. Removing all liquid is critical. It is the common cause of low yields.
+
-
            Otherwise, the pellet is loose and it is difficult to remove any liquid cell lysate.
+
-
  11.
+
-
      Pipette 50μl solution 1 to each tube.
+
-
  12.
+
-
      Resuspend the pellet by bump vortexing.
+
-
          *
+
-
            Vortex with periodic pauses. Hold for 10 seconds, take it off for 1 second, and repeat for 1 minute.
+
-
            Hold the tube horizontally to light.
+
-
            If you see any clumps of cells, keep bump vortexing. Solution 1 buffer keeps cells from lysing.
+
-
            Suspension should be homogenous for lysis reagents. Solution 1 also contains RNase for RNA digest later on.
+
-
  13.
+
-
      Check solution 2 for precipitation.
+
-
          *
+
-
            Solution 2 contains SDS detergent.
+
-
            If precipitated, heat to 55°C-66°C for 5 minutes to dissolve, cool to room temperature, and mix.
+
-
  14.
+
-
      Add 100μl solution 2 to each tube.
+
-
          *
+
-
            Alkaline detergent lyses the cell. Then, plasmid DNA is linked to form circles. RNA is digested in this step.
+
-
  15.
+
-
      Invert tubes once to mix.
+
-
          *
+
-
            Inverting more than once contaminates plasmid, causing broken chromosomal DNA to attach to plasmid DNA.
+
-
  16.
+
-
      Add 325μl solution 3 to each tube.
+
-
          *
+
-
            Solution 3 contains potassium acetate to neutralize pH. Plasmid re-natures.
+
-
  17.
+
-
      Gently invert once.
+
-
  18.
+
-
      Centrifuge (same settings)
+
-
          *
+
-
            Chromosomal DNA is pelleted.
+
-
  19.
+
-
      Remove tubes from the centrifuge.
+
-
      If the pellet is too loose or gloppy, that means the media was not completely removed and you need to start over.
+
-
      If it still looks slightly loose, try centrifuging again.
+
-
  20.
+
-
      Label 2 spin filters on the caps.
+
-
  21.
+
-
      Pour clear supernatant into spin filters and throw the tubes away.
+
-
          *
+
-
            Decant by pouring on the side away from the hinge.
+
-
  22.
+
-
      Centrifuge (same settings)
+
-
          *
+
-
            Plasmid binds to silica membrane.
+
-
  23.
+
-
      Lift the plastic filter basket from the tubes and discard liquid from the bottom of the tubes.
+
-
  24.
+
-
      Add 300μl solution 4 into the spin filters
+
-
          *
+
-
            Make sure there is ethanol in it (Our kit solution has 50% ethanol).
+
-
            Some companies don’t have ethanol, which is good for keeping the plasmid DNA bound
+
-
            to the filter as impurities are washed.
+
-
  25.
+
-
      Centrifuge (same settings)
+
-
  26.
+
-
      Lift the plastic filter basket from the tubes and discard liquid from the bottom of the tubes.
+
-
  27.
+
-
      Centrifuge (same settings) for 5 seconds
+
-
  28.
+
-
      Get another 2 2ml microcentrifuge tubes and label caps with plasmid names, sides with the date.
+
-
  29.
+
-
      Insert filter baskets in new tubes.
+
-
  30.
+
-
      Add 50μl solution 5 or double distilled water directly to the middle of the membrane.
+
-
          *
+
-
            Solution 5 has some salt, which is relatively better for subsequent storage and transformation.
+
-
            Water is better for subsequent PCR.
+
-
          *
+
-
            Plasmid DNA is eluted off the filter into the collection tube.
+
-
            (it will not stay bound when there is no salt)
+
-
  31.
+
-
      Centrifuge (same settings)
+
-
  32.
+
-
      Discard filter basket and close tube lid.
+
-
  33.
+
-
      Store plasmid DNA at -20°C. (-80°C if you want to store for several years)
+
-
  34.
+
-
      (Store kit at room temperature (20°C-25°C))
+
-
Concentrating the DNA
+
Materials:
 +
• Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
 +
• Thermocycler
-
  1.
+
Protocols:
-
      (If 50μl is too dilute for your purposes, and you want a different volume)
+
5x Phusion HF Buffer     5 ul
-
  2.
+
10 mM dNTPs               0.5 ul
-
      Add 2μl 5M NaCl and mix.
+
Oligo mixture 125 ng /250 ng /500 ng /…
-
  3.
+
Phusion DNA Polymerase               0.25 ul
-
      Add 100μl 100% cold ethanol and mix.
+
H2O                             to 25 ul
-
  4.
+
-
      Centrifuge 13,000 rpm for 5 minutes.
+
-
  5.
+
-
      Decant liquid.
+
-
  6.
+
-
      Dry ethanol in a speed vac.
+
-
  7.
+
-
      Resuspend precipitated DNA in desired volume.
+
 +
 +
98°C                        30sec
 +
40X
 +
98°C                  7 sec
 +
  70-50°C              slow ramp, 0.1°C/sec
 +
        50°C              30 sec
 +
        72°C              15 sec /kb
 +
72°C                          5 min
 +
4°C                              hold
 +
 +
 +
==<b>PCR Product Clean-up for DNA Sequencing</b>==
[[#Protocols | Back to top]]<br>
[[#Protocols | Back to top]]<br>
-
==<b>Gel Electrophoresis</b>==
+
Materials:
 +
• ExoSAP-IT® (usb, Cat. No. 78200)
 +
• Thermocycler
 +
Protocol:
 +
1. Remove ExoSAP-IT from -20°C freezer and keep on ice throughout this procedure.
 +
2. Mix 5 μl of a post-PCR reaction product with 2 μl of ExoSAP-IT for a combined 7 μl reaction volume.
 +
3. Incubate at 37°C for 15 min to degrade remaining primers and nucleotides.
 +
4. Incubate at 80°C for 15 min to inactivate ExoSAP-IT.
 +
5. The PCR product is now ready for use in DNA sequencing etc.
-
Table of Contents
 
-
    * Making the Gel
+
==<b>PCR</b>
-
    * Running the Gel
+
[[#Protocols | Back to top]]<br>
-
    * Low MW Ladder
+
-
Making the Gel
+
Materials:
 +
• Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
 +
• Thermocycler
-
    *
+
Protocols:
-
      1% agarose
+
5x Phusion HF Buffer 10 ul
-
    *
+
10 mM dNTPs               1 ul
-
      i.e. .50g agarose 50ml TBE in flask
+
DNA template   1 pg – 10 ng
-
    *
+
Forward primer (10 uM)             2.5 ul
-
      Cook in Microwave for 1 min
+
Reverse primer (10 uM)             2.5 ul
-
    *
+
Phusion DNA Polymerase             0.5 ul
-
      Let it cool down a little bit
+
H2O                         to 50 ul
-
    *
+
-
      Add 5ul of 10mg/ml Ethidium Bromide in Gel
+
-
    *
+
-
      Insert the comb. Pour out the gel and put it in the fridge
+
-
Running the Gel
 
-
    *
+
98°C    30sec
-
      5ul DNA\1ul (6X) Loading Dye into each (small) well
+
30X
-
          o
+
98°C                        10 sec
-
            (Dilute 1ul ladder with 4ul H20 and 1ul loading dye)
+
Annealing*           30 sec
-
    *
+
72°C                        15 sec /1 kb
-
      15ul DNA\3ul (6X) Loading Dye into each (large) well
+
72°C    5min
-
          o
+
4°C    hold
-
            (Dilute 1 ul ladder with 14 ul water and 3ul loading dye)
+
** Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.
-
Low MW Ladder
+
==<b>Single Colony PCR</b>
 +
[[#Protocols | Back to top]]<br>
 +
Materials:
 +
• Taq DNA Polymerase with Standard Taq Buffer (NEB, Cat. No. M0273)
 +
• 10 mM dNTP Mix (NEB, Cat. No. N0447)
 +
• Thermocycler
-
    *
+
Protocols:
-
      5ul DNA\1ul (6X) Loading Dye into each (small) well
+
10x Standard Taq Buffer                          2 ul
-
          o
+
10 mM dNTPs                     0.4 ul
-
            (Dilute .5ul ladder with 4.5ul H20 and 1ul loading dye)
+
Bacteria culture*             1 ul
-
    *
+
Forward primer (10 uM)             1 ul
-
      15ul DNA\3ul (6X) Loading Dye into each (large) well
+
Reverse primer (10 uM)             1 ul
-
          o
+
Taq DNA Polymerase                              0.2 ul
-
            (Dilute .5 ul ladder with 14.5 ul water and 3ul loading dye)
+
H2O                     to 20 ul
 +
*Bacteria culture refers to E. coli cultured in LB solution overnight.
 +
94°C    5 min
 +
25X
 +
94°C                        15 sec
 +
Annealing **        30 sec
 +
72°C                        1 min /1 kb
 +
72°C    5min
 +
4°C    hold
 +
**Anneal at Tm which is calculated with salt-adjusted method.
 +
==<b>Transformation</b>
 +
[[#Protocols | Back to top]]<br>
 +
Materials:
 +
• GC5 Chemical Competent Cells (Genesee Scientific, Cat. No. 42-653)
 +
• SOC Medium (Sigma, Cat. No. S1797)
 +
• LB Agar (Sigma, Cat. No. L3027)
 +
• Petri Dishes (VWR, Cat. No. SC25373-187)
 +
• Cell Spreader (VWR, Cat. No. 89042-018)
 +
• 37°C incubator
 +
• 37°C shaker
 +
• water bath
 +
Protocol:
 +
1. Thaw 1 tube of competent cells on ice;
 +
2. Add 3 ul of cloning product or 1-50 ng of plasmid into competent cells while stirring gently;
 +
3. Keep the tube covered by ice for 30min;
 +
4. Heat-shock the competent cells in water bath for 45 sec at 42°C;
 +
5. Put the tube on ice for 2 minutes;
 +
6. Add 450 ul of SOC medium and then put it in a 37°C shaker for 1 hour;
 +
7. Dilute and spread an appropriate amount on an LB agar plate with the appropriate antibiotics;
 +
8. Place the plate up-side-down in 37°C incubator for 16-18 hours (overnight).
-
{|align = "left"|=Materials=}
 

Revision as of 16:05, 21 October 2009

Home Project Notebook Team

Duke University's iGEM team consists of 6 undergraduate students, 2 graduate students, and 2 professors. Due to the high costs and inefficiency of the process of cloning a gene, the DUKE iGEM team has invented a new procedure which lowers costs and increases efficiency. This method, Circular Polymerase Extension Cloning* (CPEC), saves time as well, since this method does not involve ligation or restriction enzymes. The rising costs of the current method of producing biodegradable plastics has hindered its widespread use; however, this year's IGEM team has discovered a more efficient pathway to produce these biodegradable plastics. With this team's determination and motivation, they would like to present their two projects:

CPEC applied to biobricks & Biodegradable Plastic Synthesis Pathway in E. coli



*This method has been published and cited. View the paper here

What is CPEC?

Circular Polymerase Extension Cloning (CPEC) is the development of a much simplified sequence-independent cloning technology based entirely on the polymerase extension mechanism. This method extends overlapping regions between the insert and vector fragments to form a complete circular plasmid. An extremely simple theory, CPEC piggybacks PCR in splicing genes. The gene insert is modified to have ends that overlap with the ends of the linearized vector and both have similar melting temperatures. The insert and vector are placed within a PCR machine in the absence of primers. Denaturation separates the double-stranded insert and vector and the overlapping ends anneal. Polymerase extension mechanism is then used to complete the plasmid. Using this method, we are able to quickly assemble a metabolic pathway consisting of multiple enzymes and regulatory elements for the production of a biocompatible as well as biodegradable plastic polymer in E. coli.

Figure 1a. Biobrick spliced into vector using CPEC
Figure 1b. Multicomponent Biobrick system spliced into vector simulatenously using CPEC
Gel electrophoresis analysis of the final assembly of a multicomponent system after a 20-cycle CPEC


What are benefits?

The process of high-throughput cloning is bottle necked at the restriction and ligation stages. A combination of high costs, requirements for restriction site specific enzymes and general inefficiency of the process makes cloning on a large combinatorial gene library inviable. Circular Polymerase Extension Cloning (CPEC) addresses this issue by eliminating the need for restriction and ligation enzymes and thereby streamlining and condensing the procedure into the duration of 5 minutes.

Advantages/Disadvantages of BioBricks

  • BioBrick parts can be incorporated in E. coli , due to its common interphase.
  • BioBrick parts are not easily made due to its site-specific cutting of the plasmid.

Standardized CPEC

We will apply CPEC in the construction of a multi-component plasmid containing biobricks. Previous Duke iGEM projects have yielded the genes in a metabolic pathway that synthesizes poly(3HB-co-4HB), a biodegradable plastic, in E. coli. We will transform those genes into biobricks, with sticky ends, and efficiently combine them in a vector using CPEC.

Biodegradable Plastic Synthesis Pathway in E. coli

Polyhydroxyalkanoic acids (PHA), naturally occurring storage polymers found in a variety of bacteria, have received increased attention for their potential use as bioplastics that are both biodegradable and reduce reliance on petroleum-based plastics. In particular, the copolymer poly(3-hyroxybutyrate-co-4-hydroxybutyrate), or poly(3HB-co-4HB), which combines the 3HB and 4HB polymers from different bacteria (Figure 2 shows the pathway), has elastic properties ideal for a wide range of thermoplastic applications. The high cost of PHA, however, is the biggest impediment to widespread use of bioplastics. Moreover, poly(3HB-co-4HB) pathways developed so far in E. coli have yielded undesirably low and unpredictable 4HB-to-3HB ratios.

Figure 2. Pathway for poly(3HB-co-4HB) synthesis [citation]



Thus, this project aims to develop a more efficient biopathway for poly(3HB-co-4HB) while increasing the 4HB monomer composition predictably. It was hypothesized that optimizing codon permutations of the phaC gene would greatly increase affinity of PHA synthase to the 4HB monomer. To date, the phaCAB and cat2 operons have been cloned into pUC19 and PCR Blunt II-TOPO vectors for successful independent production of the 3HB and 4HB polymers (Figure 4). Ligation and transformation into E. coli as six different recombinant constructs will soon be completed and allow for engineering of the poly(3HB-co4HB) biopathway. Future directions would be to test the hypothesis to see if phaC can be manipulated to increase 4HB-to-3HB composition in poly(3HB-co-4HB) and to increase efficient production of the bioplastic by engineering the FtsZ cell division protein to allow for cells to accumulate larger quantities of PHA granules before dividing. Ultimately, once an optimal biopathway is found, the goal would be to explore a model for mass production of PHA bioplastics so that novel applications of bioplastics can be feasible economically.


Figure 3. Plasmid vector and genes used for transformation in E. coli
Figure 4. The reddish tint present on some colonies indicates the presence of PHA granules in the bacteria

Contents

Calendar

Duke University iGEM 08-09 Calendar

Below are several important milestones:

September
MTWTFSS
[http://2009.igem.org/wiki/index.php?title=Duke_University/1_September_2008&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_September_2008&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_September_2008&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_September_2008&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_September_2008&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_September_2008&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_September_2008&action=edit 7]
[http://2009.igem.org/wiki/index.php?title=Duke_University/8_September_2008&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_September_2008&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_September_2008&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_September_2008&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_September_2008&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_September_2008&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_September_2008&action=edit 14]
[http://2009.igem.org/wiki/index.php?title=Duke_University/15_September_2008&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_September_2008&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_September_2008&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_September_2008&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_September_2008&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_September_2008&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_September_2008&action=edit 21]
[http://2009.igem.org/wiki/index.php?title=Duke_University/22_September_2008&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_September_2008&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_September_2008&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_September_2008&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_September_2008&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_September_2008&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_September_2008&action=edit 28]
[http://2009.igem.org/wiki/index.php?title=Duke_University/29_September_2008&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_September_2008&action=edit 30]
October
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Duke_University/1_October_2008&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_October_2008&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_October_2008&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_October_2008&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_October_2008&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Duke_University/6_October_2008&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_October_2008&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_October_2008&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_October_2008&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_October_2008&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_October_2008&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_October_2008&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Duke_University/13_October_2008&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_October_2008&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_October_2008&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_October_2008&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_October_2008&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_October_2008&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_October_2008&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Duke_University/20_October_2008&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_October_2008&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_October_2008&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_October_2008&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_October_2008&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_October_2008&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_October_2008&action=edit 26]
[http://2009.igem.org/wiki/index.php?title=Duke_University/27_October_2008&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_October_2008&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_October_2008&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_October_2008&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_October_2008&action=edit 31]
November
MTWTFSS
          [http://2009.igem.org/wiki/index.php?title=Duke_University/1_November_2008&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_November_2008&action=edit 2]
[http://2009.igem.org/wiki/index.php?title=Duke_University/3_November_2008&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_November_2008&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_November_2008&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_November_2008&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_November_2008&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_November_2008&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_November_2008&action=edit 9]
[http://2009.igem.org/wiki/index.php?title=Duke_University/10_November_2008&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_November_2008&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_November_2008&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_November_2008&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_November_2008&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_November_2008&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_November_2008&action=edit 16]
[http://2009.igem.org/wiki/index.php?title=Duke_University/17_November_2008&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_November_2008&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_November_2008&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_November_2008&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_November_2008&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_November_2008&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_November_2008&action=edit 23]
[http://2009.igem.org/wiki/index.php?title=Duke_University/24_November_2008&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_November_2008&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_November_2008&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_November_2008&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_November_2008&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_November_2008&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_November_2008&action=edit 30]
December
MTWTFSS
[http://2009.igem.org/wiki/index.php?title=Duke_University/1_December_2008&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_December_2008&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_December_2008&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_December_2008&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_December_2008&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_December_2008&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_December_2008&action=edit 7]
[http://2009.igem.org/wiki/index.php?title=Duke_University/8_December_2008&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_December_2008&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_December_2008&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_December_2008&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_December_2008&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_December_2008&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_December_2008&action=edit 14]
[http://2009.igem.org/wiki/index.php?title=Duke_University/15_December_2008&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_December_2008&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_December_2008&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_December_2008&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_December_2008&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_December_2008&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_December_2008&action=edit 21]
[http://2009.igem.org/wiki/index.php?title=Duke_University/22_December_2008&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_December_2008&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_December_2008&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_December_2008&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_December_2008&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_December_2008&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_December_2008&action=edit 28]
[http://2009.igem.org/wiki/index.php?title=Duke_University/29_December_2008&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_December_2008&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_December_2008&action=edit 31]
January
MTWTFSS
      [http://2009.igem.org/wiki/index.php?title=Duke_University/1_January_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_January_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_January_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_January_2009&action=edit 4]
[http://2009.igem.org/wiki/index.php?title=Duke_University/5_January_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_January_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_January_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_January_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_January_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_January_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_January_2009&action=edit 11]
[http://2009.igem.org/wiki/index.php?title=Duke_University/12_January_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_January_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_January_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_January_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_January_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_January_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_January_2009&action=edit 18]
[http://2009.igem.org/wiki/index.php?title=Duke_University/19_January_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_January_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_January_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_January_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_January_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_January_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_January_2009&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Duke_University/26_January_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_January_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_January_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_January_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_January_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_January_2009&action=edit 31]
February
MTWTFSS
            [http://2009.igem.org/wiki/index.php?title=Duke_University/1_February_2009&action=edit 1]
[http://2009.igem.org/wiki/index.php?title=Duke_University/2_February_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_February_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_February_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_February_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_February_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_February_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_February_2009&action=edit 8]
[http://2009.igem.org/wiki/index.php?title=Duke_University/9_February_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_February_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_February_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_February_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_February_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_February_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_February_2009&action=edit 15]
[http://2009.igem.org/wiki/index.php?title=Duke_University/16_February_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_February_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_February_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_February_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_February_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_February_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_February_2009&action=edit 22]
[http://2009.igem.org/wiki/index.php?title=Duke_University/23_February_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_February_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_February_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_February_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_February_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_February_2009&action=edit 28]
March
MTWTFSS
            [http://2009.igem.org/wiki/index.php?title=Duke_University/1_March_2009&action=edit 1]
[http://2009.igem.org/wiki/index.php?title=Duke_University/2_March_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_March_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_March_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_March_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_March_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_March_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_March_2009&action=edit 8]
[http://2009.igem.org/wiki/index.php?title=Duke_University/9_March_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_March_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_March_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_March_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_March_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_March_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_March_2009&action=edit 15]
[http://2009.igem.org/wiki/index.php?title=Duke_University/16_March_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_March_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_March_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_March_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_March_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_March_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_March_2009&action=edit 22]
[http://2009.igem.org/wiki/index.php?title=Duke_University/23_March_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_March_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_March_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_March_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_March_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_March_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_March_2009&action=edit 29]
[http://2009.igem.org/wiki/index.php?title=Duke_University/30_March_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_March_2009&action=edit 31]
April
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Duke_University/1_April_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_April_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_April_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_April_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_April_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Duke_University/6_April_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_April_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_April_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_April_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_April_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_April_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_April_2009&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Duke_University/13_April_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_April_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_April_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_April_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_April_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_April_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_April_2009&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Duke_University/20_April_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_April_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_April_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_April_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_April_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_April_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_April_2009&action=edit 26]
[http://2009.igem.org/wiki/index.php?title=Duke_University/27_April_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_April_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_April_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_April_2009&action=edit 30]
May
MTWTFSS
        [http://2009.igem.org/wiki/index.php?title=Duke_University/1_May_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_May_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_May_2009&action=edit 3]
[http://2009.igem.org/wiki/index.php?title=Duke_University/4_May_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_May_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_May_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_May_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_May_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_May_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_May_2009&action=edit 10]
[http://2009.igem.org/wiki/index.php?title=Duke_University/11_May_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_May_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_May_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_May_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_May_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_May_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_May_2009&action=edit 17]
[http://2009.igem.org/wiki/index.php?title=Duke_University/18_May_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_May_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_May_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_May_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_May_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_May_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_May_2009&action=edit 24]
[http://2009.igem.org/wiki/index.php?title=Duke_University/25_May_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_May_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_May_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_May_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_May_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_May_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_May_2009&action=edit 31]
June
MTWTFSS
[http://2009.igem.org/wiki/index.php?title=Duke_University/1_June_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_June_2009&action=edit 2] [http://2009.igem.org/Duke_University/3_June_2009 3] [http://2009.igem.org/Duke_University/4_June_2009 4] [http://2009.igem.org/Duke_University/5_June_2009 5] [http://2009.igem.org/Duke_University/6_June_2009 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_June_2009&action=edit 7]
[http://2009.igem.org/Duke_University/8_June_2009 8] [http://2009.igem.org/Duke_University/9_June_2009 9] [http://2009.igem.org/Duke_University/10_June_2009 10] [http://2009.igem.org/Duke_University/11_June_2009 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_June_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_June_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_June_2009&action=edit 14]
[http://2009.igem.org/Duke_University/15_June_2009 15] [http://2009.igem.org/Duke_University/16_June_2009 16] [http://2009.igem.org/Duke_University/17_June_2009 17] [http://2009.igem.org/Duke_University/18_June_2009 18] [http://2009.igem.org/Duke_University/19_June_2009 19] [http://2009.igem.org/Duke_University/20_June_2009 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_June_2009&action=edit 21]
[http://2009.igem.org/Duke_University/22_June_2009 22] [http://2009.igem.org/Duke_University/23_June_2009 23] [http://2009.igem.org/Duke_University/24_June_2009 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_June_2009&action=edit 25] [http://2009.igem.org/Duke_University/26_June_2009 26] [http://2009.igem.org/Duke_University/27_June_2009 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_June_2009&action=edit 28]
[http://2009.igem.org/Duke_University/29_June_2009 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_June_2009&action=edit 30]
July
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Duke_University/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_July_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_July_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_July_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Duke_University/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_July_2009&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Duke_University/13_July_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_July_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_July_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_July_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_July_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_July_2009&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Duke_University/20_July_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_July_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_July_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_July_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_July_2009&action=edit 26]
[http://2009.igem.org/wiki/index.php?title=Duke_University/27_July_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_July_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_July_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_July_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_July_2009&action=edit 31]
August
MTWTFSS
          [http://2009.igem.org/wiki/index.php?title=Duke_University/1_August_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_August_2009&action=edit 2]
[http://2009.igem.org/wiki/index.php?title=Duke_University/3_August_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_August_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_August_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_August_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_August_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_August_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_August_2009&action=edit 9]
[http://2009.igem.org/wiki/index.php?title=Duke_University/10_August_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_August_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_August_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_August_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_August_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_August_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_August_2009&action=edit 16]
[http://2009.igem.org/wiki/index.php?title=Duke_University/17_August_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_August_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_August_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_August_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_August_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_August_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_August_2009&action=edit 23]
[http://2009.igem.org/wiki/index.php?title=Duke_University/24_August_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_August_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_August_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_August_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_August_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_August_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_August_2009&action=edit 30]
[http://2009.igem.org/wiki/index.php?title=Duke_University/31_August_2009&action=edit 31]
September
MTWTFSS
  [http://2009.igem.org/wiki/index.php?title=Duke_University/1_September_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_September_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_September_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_September_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_September_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_September_2009&action=edit 6]
[http://2009.igem.org/wiki/index.php?title=Duke_University/7_September_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_September_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_September_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_September_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_September_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_September_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_September_2009&action=edit 13]
[http://2009.igem.org/wiki/index.php?title=Duke_University/14_September_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_September_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_September_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_September_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_September_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_September_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_September_2009&action=edit 20]
[http://2009.igem.org/wiki/index.php?title=Duke_University/21_September_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_September_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_September_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_September_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_September_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_September_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_September_2009&action=edit 27]
[http://2009.igem.org/wiki/index.php?title=Duke_University/28_September_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_September_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_September_2009&action=edit 30]
October
MTWTFSS
      [http://2009.igem.org/wiki/index.php?title=Duke_University/1_October_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Duke_University/2_October_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_October_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_October_2009&action=edit 4]
[http://2009.igem.org/wiki/index.php?title=Duke_University/5_October_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_October_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_October_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_October_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Duke_University/9_October_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_October_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_October_2009&action=edit 11]
[http://2009.igem.org/wiki/index.php?title=Duke_University/12_October_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_October_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_October_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_October_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Duke_University/16_October_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_October_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_October_2009&action=edit 18]
[http://2009.igem.org/wiki/index.php?title=Duke_University/19_October_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_October_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_October_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_October_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Duke_University/23_October_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_October_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_October_2009&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Duke_University/26_October_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_October_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_October_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_October_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Duke_University/30_October_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Duke_University/31_October_2009&action=edit 31]
November
MTWTFSS
            [http://2009.igem.org/wiki/index.php?title=Duke_University/1_November_2009&action=edit 1]
[http://2009.igem.org/wiki/index.php?title=Duke_University/2_November_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Duke_University/3_November_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Duke_University/4_November_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Duke_University/5_November_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Duke_University/6_November_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Duke_University/7_November_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Duke_University/8_November_2009&action=edit 8]
[http://2009.igem.org/wiki/index.php?title=Duke_University/9_November_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Duke_University/10_November_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Duke_University/11_November_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Duke_University/12_November_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Duke_University/13_November_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Duke_University/14_November_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Duke_University/15_November_2009&action=edit 15]
[http://2009.igem.org/wiki/index.php?title=Duke_University/16_November_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Duke_University/17_November_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Duke_University/18_November_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Duke_University/19_November_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Duke_University/20_November_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Duke_University/21_November_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Duke_University/22_November_2009&action=edit 22]
[http://2009.igem.org/wiki/index.php?title=Duke_University/23_November_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Duke_University/24_November_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Duke_University/25_November_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Duke_University/26_November_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Duke_University/27_November_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Duke_University/28_November_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Duke_University/29_November_2009&action=edit 29]
[http://2009.igem.org/wiki/index.php?title=Duke_University/30_November_2009&action=edit 30]
December
MTWTFSS
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Protocols

CPEC Cloning

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CPEC cloning Materials: • Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553) • Thermocycler

Protocol: 5x Phusion HF Buffer 4 ul 10 mM dNTPs 0.4 ul Vector 50 ng/1kb Insert x ng* Phusion DNA Polymerase 0.2 ul H2O to 20 ul

  • The amount of insert is determined so that the molar ratio for vector and insert is 1 to 2.

98°C 30sec 10X 98°C 10 sec Annealing** 30 sec 72°C x sec*** 72°C 5min 4°C hold

    • Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.
      • The extension time is usually calculated according to the shortest piece with 15 sec /kb if the cloning is not complicated. For example, if there is only one insert and is shorter than the vector, say, 600 bp, then I will use 15 sec for extension. Refer to the published paper for detailed information.


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DNA Purification

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DNA purification Materials : • E.Z.N.A Gel Purification Kit (Omega Bio-Tek, Cat No. D2500-02 ) • Water bath equilibrated to 55-65C • Microcentrifuge capable of at least 10,000 x g • Nuclease-free 1.5 ml centrifuge bottles • Absolute (95%-100%) ethanol • Protective eye-wear • Isopropanol (for fragments < 500 bp only) Protocol: 1. Perform agarose gel electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended, however, that fresh TAE buffer or TBE buffer be used as running buffer. Do not re-use running buffer as its pH will increase and reduce yields. 2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean scalpel. 3. Determine the approximate volume of the gel slice by weighing it in a clean 1.5 ml microfuge tube. Assuming a density of 1 g/ml of gel, the volume of gel is derived as follows: A gel slice of mass 0.3 g will have a volume of 0.3 ml. Add equal volume of Binding Buffer (XP2). Incubate the mixture at 55C-60C for 7 min or until the gel has completely melted. Mix by shaking or inverting the tube every 2-3 minutes. Centrifuge the tube briefly to collect all the liquid to the bottom of the tube. Note: For DNA fragment less than 500bp, add 1 sample volume of isopropanol after the addition of Binding Buffer (XP2). 4. Apply up to 700 ul of the DNA/agarose solution to a HiBind® DNA spin column assembled in a clean 2 ml collection tube (provided) and centrifuge in a microcentrifuge at 8,000-10,000 x g for 1 min at room temperature. Discard the liquid. Re-use the collection tube in Steps 5-8. For volumes greater than 700 ul, load the column and centrifuge successively, 700 ul at a time. Each HiBind® spin-column has a total capacity of 25-30 ug DNA. 5. Discard liquid and add 300ul Binding Buffer. Centrifuge at 10,000 x g for 1 minutes. 6. Add 700 ul of SPW Buffer diluted with absolute ethanol into the column and wait 2-3 minutes. Centrifuge at 10,000 x g for 1 min at room temperature to wash the sample. 7. Discard liquid and repeat step 6 with another 700 ul SPW Buffer. 8. Discard liquid and, re-using the collection tube, centrifuge the empty column for 1 min at maxi speed (>13,000 x g) to dry the column matrix. This drying step is critical for good DNA yields. 9. Place column into a clean 1.5 ml microcentrifuge tube (not provided). Add 30-50 ul depending on desired concentration of final product) Elution Buffer (or sterile deionized water) directly to the center of the column matrix, then incubate for 1 minute. Centrifuge 1 min at maxi speed (>13,000 x g) to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lo


PCA (Polymerase Cycle Assembly

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Materials: • Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553) • Thermocycler

Protocols: 5x Phusion HF Buffer 5 ul 10 mM dNTPs 0.5 ul Oligo mixture 125 ng /250 ng /500 ng /… Phusion DNA Polymerase 0.25 ul H2O to 25 ul


98°C 30sec 40X 98°C 7 sec 

  70-50°C               slow ramp, 0.1°C/sec
        50°C               30 sec 
        72°C               15 sec /kb

72°C 5 min 4°C hold


PCR Product Clean-up for DNA Sequencing

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Materials: • ExoSAP-IT® (usb, Cat. No. 78200) • Thermocycler Protocol: 1. Remove ExoSAP-IT from -20°C freezer and keep on ice throughout this procedure. 2. Mix 5 μl of a post-PCR reaction product with 2 μl of ExoSAP-IT for a combined 7 μl reaction volume. 3. Incubate at 37°C for 15 min to degrade remaining primers and nucleotides. 4. Incubate at 80°C for 15 min to inactivate ExoSAP-IT. 5. The PCR product is now ready for use in DNA sequencing etc.


==PCR Back to top

Materials: • Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553) • Thermocycler

Protocols: 5x Phusion HF Buffer 10 ul 10 mM dNTPs 1 ul DNA template 1 pg – 10 ng Forward primer (10 uM) 2.5 ul Reverse primer (10 uM) 2.5 ul Phusion DNA Polymerase 0.5 ul H2O to 50 ul


98°C 30sec 30X 98°C 10 sec Annealing* 30 sec 72°C 15 sec /1 kb 72°C 5min 4°C hold

    • Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.

==Single Colony PCR Back to top
Materials: • Taq DNA Polymerase with Standard Taq Buffer (NEB, Cat. No. M0273) • 10 mM dNTP Mix (NEB, Cat. No. N0447) • Thermocycler

Protocols: 10x Standard Taq Buffer 2 ul 10 mM dNTPs 0.4 ul Bacteria culture* 1 ul Forward primer (10 uM) 1 ul Reverse primer (10 uM) 1 ul Taq DNA Polymerase 0.2 ul H2O to 20 ul

  • Bacteria culture refers to E. coli cultured in LB solution overnight.

94°C 5 min 25X 94°C 15 sec Annealing ** 30 sec 72°C 1 min /1 kb 72°C 5min 4°C hold

    • Anneal at Tm which is calculated with salt-adjusted method.

==Transformation Back to top

Materials: • GC5 Chemical Competent Cells (Genesee Scientific, Cat. No. 42-653) • SOC Medium (Sigma, Cat. No. S1797) • LB Agar (Sigma, Cat. No. L3027) • Petri Dishes (VWR, Cat. No. SC25373-187) • Cell Spreader (VWR, Cat. No. 89042-018) • 37°C incubator • 37°C shaker • water bath Protocol: 1. Thaw 1 tube of competent cells on ice; 2. Add 3 ul of cloning product or 1-50 ng of plasmid into competent cells while stirring gently; 3. Keep the tube covered by ice for 30min; 4. Heat-shock the competent cells in water bath for 45 sec at 42°C; 5. Put the tube on ice for 2 minutes; 6. Add 450 ul of SOC medium and then put it in a 37°C shaker for 1 hour; 7. Dilute and spread an appropriate amount on an LB agar plate with the appropriate antibiotics; 8. Place the plate up-side-down in 37°C incubator for 16-18 hours (overnight).




6/3/09
Experiment: PCR with contents of tube labeled 7 as DNA template, Gel with 100 bp ladder to check PCR
Results: Incorrect ladder and template
Conclusion: Should use 1 kb ladder, Need to find correct template

6/4/09
E: Gel to check Bioplastics #1-5
R: All correct sized bands
C: Use one as template for PCR

E: PCR with Bioplastics #5 as template

5x HF buffer 5 ul
dNTPs 2 ul
forward primer 1.25 ul
reverse primer 1.25 ul
DNA template (plasmid) 0.5 ul
Phusion DNA polymerase 0.3 ul
H2O 14.7 ul
25 ul

98°C 30s 1x
98°C 15s
55°C 30s
72°C 1m 10s 35x
72°C 5m
4°C infinity 1x

Fragment # Name Size/Length
1 pASKphaA 4.2 kb
2 phaB 900 bp
3 Termcat2 1.7 kb
4 phaC 1.9 kb

6/5/09
E: Gel to check PCR
R: Correct band for fragments 2 and 4, No band for fragments 1 and 3
C: Cut correct bands out of gel, Try PCR with another template, different conditions

E: PCR with Bioplastics #1 as template, increased extension time to 1m 20s

6/6/09
R: Correct band for fragments 1 (weak) and 2, No band for fragments 3 and 4
C: Cut out correct bands, Do PCR again with same template for fragments 1 and 2, Try PCR with different template for fragments 3 and 4

E: PCR with Bioplastics #1 (fragments 1 and 2) and #3 (fragments 3 and 4) as templates


6/8/09
E: Gel to check PCR, Gel with leftover PCR of right sized fragments
R: Correct bands for all fragments
C: Cut out bands

6/9/09
Gel Extraction
1. Weigh cut gel piece(s). Add at least 1 ul binding buffer per mg of gel.
2. Place tube at 50-60°C for 10-15 min or until gel is completely dissolved. Vortex every few minutes.
3. Add solution to spin column. Centrifuge at max speed for 30s. Discard filtrate.
4. Add 300 ul binding buffer to column. Centrifuge for 30s. Discard filtrate.
5. Add 500 ul wash buffer. Centrifuge for 30s. Discard filtrate.
6. Repeat step 5.
7. Centrifuge tubes for at least 2 min. Discard collection tube.
8. Obtain 1.7 ml centrifuge tube. Add 40-50 ul elution buffer to spin column. Centrifuge for 1 min. Discard spin column.

E: Measured concentration of DNA fragments
R: Very low concentrations of fragments 1 and 3
C: Do PCR with fragments as template, greater volume to increase amount

E: PCR with fragments 1 and 3 as templates

6/10/09
E: Gel to check PCR
R: Smears at bottom of gel
C: Should use Bioplastics #1 as template to increase amount of fragment 1 and Bioplastics #3 as template to increase amount of fragment

E: PCR with Bioplastics #1 and #3 as templates and doubled volume, Gel to obtain more of fragments
R: Smears for fragment 1, Correct bands for fragment 2, Correct bands and smears for fragment 3
C: PCR with different conditions

E: PCR with Bioplastics #3 as template, extension time increased to 1m 30s, cycles increased to 40

6/11/09
E: Gels to check PCR
R: Correct bands for fragment 1, 2 and 4 and possibly correct band for fragment 3
C: Cut out bands

E: Measured concentration of DNA fragments
R: Still low concentrations of fragments 1 and 3
C: Do more PCRs to obtain more of fragments 1 and 3


6/15/09
E: PCRs with Bioplastics #1 (fragments 1 and 2) and #3 (fragments 3 and 4) as templates, doubled volume, eliminated annealing step, varied extension (plus annealing) time based on fragment size, Gels to check PCRs

5x HF buffer 10 ul
dNTPs 4 ul
forward primer 2.5 ul
reverse primer 2.5 ul
DNA template (plasmid) 1 ul
Phusion DNA polymerase 0.8 ul
H2O 29.2 ul
50 ul

98°C 30s 1x
98°C 15s
72°C varies 35x
72°C 5m
4°C infinity 1x

Annealing Time: 30s
Extension Time: 15 s * size/length (kb)

Fragment # Name Size/Length (bp) Time (s)
1 pASKphaA 4215 100
2 phaB 896 50
3 Termcat2 1701 60
4 phaC 1872 65

R: Bands for fragments 1 and 2, No bands for fragments 3 and 4
C: Cut out bands of fragments 1 and 2, PCR again to obtain fragments 3 and 4

6/16/09
E: PCR with Bioplastics #2 as template and Phusion mix (done by Maggie), Gels to check PCRs

2x Phusion mix 25 ul
forward primer 2.5 ul
reverse primer 2.5 ul
DNA template (plasmid) 1 ul
H2O 19 ul
50 ul

R: No bands for fragments 3, Bands for fragment 4
C: Cut out bands of fragment 4, PCR again to obtain fragment 3

E: Measured concentration of DNA fragments 1 and 2
R: Concentrations okay
C: Do assembly PCR once enough of fragments 3 and 4 obtained

6/17/09
E: PCR with Bioplastics #4 as template, using Phusion enzyme protocol (shorter denaturation time, lower annealing temperature), Gel to check PCRs

98°C 15s 1x
98°C 1s
50°C 5s
72°C 30s 35x
72°C 2m
4°C infinity 1x

R: Correct bands
C: Cut out bands

E: Measured concentration of DNA fragments 3 and 4
R: Concentrations high
C: Do assembly PCR


6/18/09
E: Assembly PCR, Gels to check PCRs

2x Phusion mix 12.5 ul
insert 7.14 ul
H2O 5.36 ul
25 ul

98°C 30s 1x
98°C 10s
70-55°C 5s
72°C 30s 25x
72°C 2m
4°C infinity 1x

Fragment # Name Size/Length (bp) Concentration (ng/ul) Mass (ng) Volume (ul)
1 pASKphaA 4215 41 194 4.73
2 phaB 896 68 41 0.60
3 Termcat2 1701 69 78 1.13
4 phaC 1872 126 86 0.68
~400 7.14

Length of plasmid: 8360 bp + 4 Histags * 6 aa * 3 bp = 8432 bp

R: Smear because of misformed gel, no band
C: Change conditions

6/19/09
E: Assembly PCR (done by Maggie) with different conditions (decreased extension time, increased number of cycles)

5x HF buffer 5 ul
dNTPs 2 ul
insert 7.18 ul
Phusion DNA polymerase 0.4 ul
H2O 10.42 ul
25 ul

98°C 30s 1x
98°C 10s
70-55°C 30s
72°C 2m 40x
72°C 5m
4°C infinity 1x

Fragment # Name Mass (ng) Volume (ul)
1 pASKphaA 195.3 4.76
2 phaB 41 0.60
3 Termcat2 78.4 1.14
4 phaC 86.4 0.68
~400 7.14
6/20/09
E: Gel to check PCR
R: Correct band (faint)
C: Low efficiency, Use different method (Infusion kit or PCR with 2 fragments first)

6/22/09
LB Agar Plates with Chlorophenicol (CAM), anhydrotetracycline, Nile Red (NR)
stock plate
CAM 50 mg/ul 20 ug/ml
anhyd 2 mg/ml 150 ng/ml
NR 0.25 mg/ml 0.5 ug/ml

E: PCR with fragments 1 and 2 and different conditions (decreased annealing and extension times and number of cycles), Gel to check PCR

2x Phusion mix 12.5 ul
pASKphaA 4.76 ul
phaB 0.60 ul
H2O 7.14 ul
25 ul

98°C 30s 1x
98°C 10s
70-55°C 10s
72°C 1m 10s 20x
72°C 5m
4°C infinity 1x

R: No band/smear
C: Change conditions

6/23/09
E: Assembly PCR with different conditions (no slow ramp)

6/24/09
E: Gel to check PCR
R: Correct band (faint)
C: Do transformation with competent cells


6/24/09
Transformation with High Efficiency GC5 Competent Cells
1. Remove competent cells from -70°C and place on ice. Thaw for 5-10 min.
2. Gently mix cells by tapping tube.
3. Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix.
4. Place tubes on ice for 30 min.
5. Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake!
6. Add 450 ul RT SOC Medium to each transformation reaction.
7. Incubate at 37°C for 1 hr with shaking at 225-250 rpm.
8. Spread on LB agar plates containing appropriate antibiotic.
9. Incubate plates at 37°C overnight (12-16 hrs).

6/26/09
Pick a colony from a streaked selective plate to inoculate 10 ml of LB medium supplemented with the appropriate selection antibiotic. Incubate 12-18 hrs at 37°C while shaking at 200-250 rpm.

6/27/09
Harvest bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for 2 min at RT. Decant supernatant and remove remaining medium.

Purification/Mini prep
1. Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or pipet up and down until no cell clumps remain.
2. Add 250 Lysis Solution and mix thoroughly by inverting tube 4-6 times until solution becomes viscous and slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 ul Neutralization Solution and mix thoroughly by inverting tube 4-6 times.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer supernatant to spin column by decanting or pipetting. Avoid disturbing or transferring white precipitate.
6. Centrifuge for 1 min. Discard flow-through and replace column in collection tube.
7. Add 500 ul Wash Solution to spin column. Centrifuge for 30-60s and discard flow-through.
8. Repeat step 7.
9. Centrifuge for 1 min to remove residual Wash Solution.
10. Transfer spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to center of spin column membrane. Do not touch pipette tip to membrane. Incubate 2 min at RT and centrifuge for 2 min.
Note: An additional elution step with Elution buffer or water will recover residual DNA from the membrane and increase overall yield by 10-20%.
11. Discard column and store purified plasmid DNA at -20°C.

6/29/09
E: Restriction digests, Gel to check digests

Digest 1
NdeI (FastDigest) 1 ul
SpeI (NEB) 1 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Sizes/Lengths: 5 kb, 3 kb, 460 bp, (80bp)

Digest 2 (Did not do)
NdeI (FastDigest) 2 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Sizes/Lengths: 5.4 kb, 3 kb

Digest 3
BamHI (FastDigest) 1 ul
XhoI (FastDigest) 1 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Sizes/Lengths: 6.6 kb, 1.8 kb

Digest 4
BamHI (FastDigest) 2 ul
10x FastDigest buffer 2 ul
plasmid 1 ul (~100 ng)
H2O 15 ul
20 ul
Band Size/Length: 8.4 kb

R: Correct bands and sizes for plasmid 2 and digests 1, 3, and 4, correct band and size for plasmid 4
C: Do more tests on plasmid 2 such as sequencing to determine if plasmid is carryover, Try different primers

 



Advisors

TianJ.jpg Dr. Jingdong Tian
jtian(at)duke.edu
Duke BME Department & Duke IGSP 		YouL.jpg Dr. Lingchong You
you(at)duke.edu
Duke BME Department & Duke IGSP 		Yuanf.jpg Dr. Fan Yuan
fyuan(at)duke.edu Duke BME Department

Graduate Students

Quanm.jpg Maggie Jiayuan Quan
jq7(at)duke.edu
Graduate Student 		Rezaf.jpg Faisal Reza
faisal.reza(at)duke.edu
Graduate Student

Students

Anga.jpg Andrew Ang
andrew.ang(at)duke.edu
Andrew Ang is a freshman at Duke, majoring in Biomedical Engineering. Apart from class and iGEM, he is involved in the Jazz Ensembles program and Asian Students Association at Duke. His hobbies include piano, saxophone, tennis, and squash. He is interested in molecular and synthetic biology, biomolecular engineering and medical research. He has previously worked as part of the MIT 2008 team, and he is excited to participate in iGEM again this year, and many more years to come.
Chienk.jpg Kevin Chien
kevin.chien(at)duke.edu
Fuy.jpg Yaoyao Fu
yf21(at)duke.edu
Faith.jpg Faith Kung
fk8(at)duke.edu

Faith Kung is a senior at Duke majoring in Biomedical Engineering with minors in Music and Biology. She enjoys working in a lab. Besides academics, her hobbies include arts and crafts, dance, and figure skating. Also, she is actively involved in the IV Christian Fellowship. Faith is applying to PhD programs in Biomedical Science and hopes to pursue a career in scientific research and education. She is excited about attending the iGEM competition this year.

PrasadaS.jpg Sahil Prasada
sahil.prasada(at)duke.edu

Sahil Prasada is a freshman at Duke. He plans to pursue medicine as a career. His interests lie in Detroit sports, tennis, and dancing. He is a member of the DBS Raas team on campus. He is currently in the Trinity School of the Arts and Sciences but is considering transferring to the Pratt School of Engineering. He hopes that the Detroit Lions may one day win the Superbowl. While waiting for this to occur, he will attend the iGEM competition and is looking forward to winning an award.

Nicholas.jpg Nicholas Tang
nicholas.tang(at)duke.edu
Zhup.jpg Peter Zhu
peter.zhu(at)duke.edu
Peter Zhu Is a freshman at Duke University and a North Carolina local. Though he's not sure yet what to do with his life, he thinks Biomedical Engineering and pre-Law is looking pretty good. When he's not busy with the routines of life, he is listening to the Billboard Top 100, playing Chopin Preludes, searching for new places to eat, playing tennis, studying poker, and gaming Starcraft/DoTA. Peter is a regular at Bail Hai Mongolian Grill, Lime and Basil Vietnamese Pho, and Five Guy's Burgers---bacon cheeseburger with all toppings of course.

Sponsors

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