Team:Tsinghua/Protocol

From 2009.igem.org

Home Background Brainstorming Design Experiment Results Conclusion Protocol


Contents

Microbiological Culture

General Protocol

Essential requirements for growth

1) supply of suitable nutrients

2) source of energy

3) water

4) appropriate temperature

5) appropriate pH

6) appropriate levels (or absence) of oxygen

1x LB medium (Luria-Bertani medium)

tryptone, 10 g

yeast extract, 5 g

NaCl, 10 g

deionized H2O, to 950 ml

Adjust the pH to 7.0 with 5 N NaOH. Adjust the volume to 1 liter with H2O. Sterilize by autoclaving.

Tips

1) Remember to do PARALLEL experiments at the same time!

2) Bacteria-free technique is a must in the wetlab, especially for the induction of protein expression, and the identification of positive clone.

3) Bear in mind that bacteriophage is just nearby!

4) DO NOT add antio-biotics when recombination bacteria are first picked out and transplanted from a plate to a culture bottle.

Molecular Cloning

Isolation of Plasmid DNA

General Protocol

1)Bacteria culture: Inoculate the single colony harboring plasmid into 20 ml LB medium containing antibiotics, 37℃, 12-16 hours.

2)Pellet 1–3 ml of cells by centrifugation for 1–2 minutes at 12000rpm. Decant the supernatant. (1.4ml each time and collect about 3 ml culture of E.coli in 1.5ml eppendorf tube.) Completely resuspend the cell pellet in 100 μl solution 1 by vigorous vortex.

3)Add 150 μl solution 2, mix by inverting the tube 4-6 times gently and incubate the tube on ice for 1-2 minutes (do not exceed this period!). The cell suspension should be clear immediately.

4)Add 150 μl solution 3, invert the tube gently several times, place the tube at room temperature for 5 minutes, and centrifuge at 12000rpm for 15 minutes.

5)Add 420 μl binding buffer to the mini-spin column. Then transfer the supernatant of procedure 3 to the same mini-spin column. Mix the supernatant and binding buffer with pipette carefully. Then place the column in another tube, centrifuge at 12000rpm for 30 and then discard waste liquid in the tube.

6)Add 750 μl wash buffer to the column, and centrifuge at 12000rpm for 1minute.

7)Repeat procedure 5. Then centrifuge at 12000rpm for 2minutes. Eliminate wash buffer as thoroughly as possible. The ethanol in wash buffer will impact the following enzyme-catalyzed reactions.

8)Carefully move the column into another clean tube. Add 50 μl Elution buffer or water into the column, place it at room temperature for 5minutes, andcentrifuge at 12000rpm for 1 minute.

Tips

1)Elution buffer should be added in the middle of adsorption material to guarantee all the plasmid DNA recovered.

2)To increase the recovery efficiency, increase the elution volume or elution times if it is necessary

3)0.5ml RNaseA(10mg/ml)can be added to the purified plasmid to eliminate RNA thoroughly.

4)If the molecular weigh of purified plasmid exceeds 10kb,the column may be placed in 70℃ water bath for 3-5 minutes to ensure plasmid DNA totally recovered.

Transformation of Recombinant DNA

General Protocol

1)Pipette competent cells suspension into the tubes, 100 μl each tube (If the competent cells are taken from -70℃, perform following steps immediately after thawing).

2)Add 10 μl of recombinant plasmid into the tube which is the tube with competent cells.

3)Mix the solutions gently, keep on ice for 20-30 minutes.

4)Heat shock by transferring the tubes to a water bath of 42℃ for 1 to 2 minutes.

5)Immediately return the tube to the ice bath. Keep on ice for 2 minutes.

6)Add 0.9 ml of LB (with no antibiotics added) into each tube. Incubate the tubes for 45 minutes to 1 hour at 37℃ to allow the cells to express their antibiotic gene product.

7)Spread about 200 μl of the resulting solutions (do dilution if necessary) on LB plates (with corresponding antibiotic added). After complete absorption of liquid LB, upside down the plates and incubate the plates at 37℃ overnight.

Tips

1)Never spread the transformation solution until you have assured that the glass stick is cooled down!

Ethanol Precipitation

General Protocol

1)Add 2 volume of 100% ethanol and 1/10 volume of 3M NaAc (pH=5.2) to the enzymatic digested solution.

2)Place the tube at -20℃ refrigerator for 20min.

3)Centrifuge at 4℃, 12,000rpm for 10 min.

4)Carefully discard the supernatant and add 100μl 70% ethanol.

5)Centrifuge and discard the supernatant again.

6)Desiccation at room temperature for 15 minutes.

7)Add 10 μl distilled water to dissolve DNA.

Tips

PCR

General Protocol

1. Standard PCR

PCR System
Reagent Concentration/Activity Volume (50uL System) Volume (100uL System)
10x Pyrobest buffer II 10x 5 10
Pyrobest 0.3 0.5
dNTPmix 10mM each 1 2
Primer 1 10uM 1 2
Primer 2 10um 1 2
Template DNA changeable 0.5 1
MgCl2(Deletable) 0.2M 0.5 1
ddH2O 40.5 81

(Pyrobest DNA polymerase from Takara Co.Ltd.)


PCR Program
Step Condition Time
1 95℃ 5min
2 95℃ 30sec
3 [Tm(fu)-4]℃ 30sec
4 72℃ DNA length/kb/min
5 RETURN TO STEP 2 30-35 cycles
6 72℃ 10min
7 4℃ HOLD



2. Fusion PCR


The basic system is similar to common PCR. There are some notes to raise the fusion efficiency:

a. Complementary region length: 15-20bp

b. Raise the annealing temperature in the fusion step.


Fusion PCR Program
Step Condition Time
1 95℃ 5min
2 95℃ 30-50sec
3 {Tm(fu)+[(-2)~5]}℃ 40-80sec
4 72℃ DNA length/kb/min
5 RETURN TO STEP 2 10-15 cycles
6 72℃ 5min
7 Add amplification Primers
8 95℃ 2-5min
9 95℃ 30sec
10 [Tm(fu)-4]℃ 30sec
11 72℃ DNA length/kb/min
12 RETURN TO STEP 2 25-30 cycles
13 72℃ 10min
14 4℃ HOLD

Tips

Protein Isolation and Identification

Protein Isolation for prokaryotes

1) Bacteria of positive clone are grown in sterilized medium containing 1% anti-biotics at 37 °C.

2) At OD600 = 0.8, or overnight, cells are induced by addition of IPTG and left at 25 °C overnight.

3) The suspension is then centrifuged (30 min, 4500 rpm, 6 °C) and the pellet resuspended in lysis buffer (20 mM imidazol, 0.25M NaCl) and the cell suspension sonicated two cycles, using an ultrasonic processor. For each cycle, the working time/ pausing time is 4s/ 6s.

4) The cells are pelleted by centrifugation at 12000rpm for 50 min at 10 °C, using a high-speed centrifuge.

5) The supernatant is loaded on a Ni-NTA column, washed with lysis buffer and eluted with Buffer B (400 mM imidazol).

6) Freeze the fractions at the temperature of -20 °C, for other detections.

Protein Isolation for eukaryotes

1) The cells are pelleted by centrifugation at 800rpm for 5 min at 4 °C. And wash with ice-cold PBS twice.

2) Lysis with TEN-T buffer at 4 °C for 1 hour. And then centrifuge at 12000rpm for 15 min at 4 °C.

3) The suspension is kept at the temperature of -20 °C for other detection, while the pellet is discarded.

Protein Identification ( SDS-PAGE)

Pouring the separation gel

1) Assemble gel sandwich according to the manufacturer's instructions, or according to the usage of alternative systems. For Mini-Gel, be sure that the bottom of both gel plates and spacers are perfectly flush against a flat surface before tightening clamp assembly. A slight misalignment will result in a leak.

2) Combine solutions C and B and water in a small Erlenmeyer flask or test tube.

3) Add ammonium persulfate and TEMED, and mix by swirling or inverting container gently (excessive aeration will interfere with polymerization). Work rapidly at this point because polymerization will be under way.

4) Carefully introduce solution into gel sandwich using a pipet. Pipet solution so that it descends along a spacer. This minimizes the possibility of air bubbles becoming trapped with the gel.

5) When the appropriate amount of separating gel solution has been added (in the case of the Mini-Gel, about 1.5cm from top of front plate or 0.5cm below level where teeth of comb will reach), gently layer about 1cm of water on top of the separating gel solution. This keeps the gel surface flat.

6) Allow gel to polymerize (30-60 min). When the gel has polymerized, a distinct interface will appear between the separating gel and the water, and the gel mold can be tilted to verify polymerization.

Pouring the stacking gel

1) Pour off water covering the separating gel. The small droplets remaining will not disturb the stacking gel.

2) Combine Solution C and B and water in a small Erlenmeyer flask or a test tube.

3) Add ammonium persulfate and TEMED and mix by gently swirling or inverting the container.

4) Pipet stacking gel solution onto separating gel until solution reaches top of front plate.

5) Carefully insert comb into gel sandwich until bottom of teeth reach top of front plate. Be sure no bubbles are trapped on ends of teeth. Tilting the comb at a slight angle is helpful for insertion without trapping air bubbles.

6) Allow stacking gel to polymerize (about 30 min).

7) After stacking gel has polymerized, remove comb carefully (making sure not to tear the well ears).

8) Place gel into electrophoresis chamber.

9) Add electrophoresis buffer to inner and outer reservoir, making sure that both top and bottom of gel are immersed in buffer.

Protein sample preparation

Protein sample is diluted with an equal volume of sample buffer. In the experiment, take 10ul samples and 10ul sample buffer. The mixture is heated for 5 min at 100℃. Introduce sample solution into well using a Hamilton syringe. Layer protein solution on bottom of well and raise syringe tip as dye level rises. Be careful to avoid introducing air bubbles as this may allow some of sample to be carried to adjacent well. Rinse syringe thoroughly with electrode buffer or water before loading different samples. Include molecular weight standards in one or both outside wells. A slab gel is especially useful for molecular weight determinations since the sample and molecular weight standard proteins can be run under identical conditions on a single gel. There are a number of commercially available SDS-PAGE molecular weight standards which give a good spread of molecular weight lines in a gel.

Running a gel

1) Attach electrode plugs to proper electrodes. Current should flow towards the anode.

2) Turn on power supply to 200V.

3) The dye front should migrate to 1cm from the bottom of the gel in 30-40 min for two 0.75mm gels (40-50 min for 1.5mm gels).

4) The high electrical current used in gel electrophoresis is very dangerous. Never disconnect electrodes before first turning off the power source. If using an electrophoresis apparatus, which is not completely shielded from the environment, always leave a clearly visible sign warning that electrophoresis is in progress.

5) Turn off power supply.

6) Remove electrode plugs from electrodes.

7) Remove gel plates from electrode assembly.

8) Carefully remove a spacer, and inserting the spacer in one corner between the plates, gently pry apart the gel plates. The gel will stick to one of the plates.

Stain and destain the gel

1) Stain the gel in staining solution for overnight at RT.

2) Destain the gel, change fresh destaining solution several times.

Reagens

BUFFER SYSTEM

(A) Lower buffer: 18.17g Tris, 0.4g SDS, pH 8.8 (HCl), added H2O to 100ml (1.5mol/L Tris-HCl buffer).

(B) Upper buffer: 6.06g Tris, 0.4g SDS, pH 6.8 (HCl), added H2O to 100ml (0.5mol/L Tris-HCl buffer).

(C) 30% Acrylamide: 30g Acrylamide, 0.8g Bisacrylamide, added H2O to 100ml.

(D) 10%(w/v) Ammonium persulfate (fresh): 0.1g ammonium persulfate, added H2O to 1ml.

Electrophoresis Buffer: 3g Tris, 14.4g glycine, 1g SDS, added H2O to 1 liter H2O, Distilled water.

Sample buffer: 1g SDS, 5ml Glycerol, 50mg Bromophenol blue, 2.5ml Mercaptoethanol, 5ml Upper buffer, added H2O to 50ml.

Staining and destaining solution

Staining solution:

0.25g Coomassie Blue R-250 was dissolved in 50ml methanol, added 8ml acetic acid and 42ml H2O to 100ml.

Destaining solution:

200ml methanol, 70ml acetic acid, added H2O to 1000ml.

Separation Gel Preparation

Table 1 12.5% Running gel (Separating gel)
Reagen Volumn
A 4.5ml
C 7.5ml
H2O 5.9ml
TEMED 0.02ml
D(fresh) 0.07ml
total 18ml

Stacking Gel Preparation

Table 2 3% Stacking gel
Reagen Volumn
B 2.0ml
C 0.8ml
H2O 5.2ml
TEMED 0.01ml
D(fresh) 0.04ml
total 8.05ml

Cell Culture

General protocol

HeLa cells frozen in liquid nitrogen are revived in routine method, and inoculated in RPMI-1640 blended with 10% fetus cattle serum, cultured in six-well culture plate. The plates are put in the incubator with the condition of 37℃, 5%CO2. The growth of cells is observed every day. 70% of the cells confluence is digested with EDTA and then is made into cell suspension.

Plasmid DNA Transfection

Use the following procedure to transfect DNA into mammalian cells in a 6-well format. All amounts and volumes are given on a per well basis. Prepare complexes using a DNA (μg) to Lipofectamine™ 2000 (μl) ratio of 1:2 to 1:3 for most cell lines. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity.

1) Adherent cells: One day before transfection, plate 0.5-2 x 105 cells in 500 μl of growth medium without antibiotics so that cells will be 90-95% confluent at the time of transfection.

Suspension cells: Just prior to preparing complexes, plate 4-8 x 105 cells in 500 μl of growth medium without antibiotics.

2) For each transfection sample, prepare complexes as follows:

a. Dilute DNA in 50 μl of Opti-MEM® I Reduced Serum Medium without serum (or other medium without serum). Mix gently.

b. Mix Lipofectamine™ 2000 gently before use, then dilute the appropriate amount in 50 μl of Opti-MEM® I Medium. Incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.

c. After the 5 minute incubation, combine the diluted DNA with diluted Lipofectamine™ 2000 (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy). Note: Complexes are stable for 6 hours at room temperature.

3) Add the 100 μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.

4) Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for transgene expression. Medium may be changed after 4-6 hours.

5) For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection.

Add selective medium (if desired) the following day. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as DNA and Lipofectamine™ 2000 concentrations. Make sure that cells are greater than 90% confluent and vary DNA (μg): Lipofectamine™ 2000 (μl) ratios from 1:0.5 to 1:5.

Tips

1) Do not add antibiotics to media during transfection as this causes cell death.

2) Maintain the same seeding conditions between experiments.

3) Remember to test serum-free media for compatibility with Lipofectamine™ 2000 since some serum-free formulations may inhibit cationic lipid-mediated transfection.

Reference

Molecular Biology Section

[1] Sambrook J, Maniatis T, Fritsch EF. Molecular Cloning: a Laboratory Manual. cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 3rd ed., 2001.

[2] Robert F. Weaver. Molecular Biology, McGrawHill, 4th edition, 2007.

[3] http://2008.igem.org/Team:Tsinghua/Notebook

Biochemistry Section

[1] Bingbin Yu, Mingxing Duan, Jiangang Chen. Techniques of Biochemical Experiments, School of Life Sciences in Tsinghua, 2000

[2] David L. Nelson, Michael M. Cox. Lehninger PRINCIPLES OF BIOCHEMISTRY, fourth edition, 2004

Cell Biology Section

[1] Invitrogen Lipofectamine™ 2000 Protocol

[2] Zhenqiang Situ. Cell Culture. World Book Publishing Company, 2007