Team:Newcastle/Labwork/14 September 2009

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(→Ligating the cotC BioBrick with pMUTIN4)
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{{:Team:Newcastle/Left}}
{{:Team:Newcastle/Left}}
__NOTOC__
__NOTOC__
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=Lab Work - 14/09/09=
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[[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]]
-
==Metal Sensing team==
+
=Formal Lab Session - 14th September 2009=
 +
[[Image:Team Newcastle 2009 iGEM 14-09-09 IMG 1131.JPG|300px|center]]
 +
<br>
 +
=<font color="Orange"><u>Overview</u></font>=
 +
<font color="Orange">
 +
*[[#Metal Sensor Team|Metal Sensor Team]] '''- '''
 +
<br>
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*[[#Sporulation Tuning/Chassis Team|Sporulation Tuning/Chassis Team]] '''- '''
 +
<br>
 +
</font>
 +
<br>
 +
==<u>Metal Sensor team</u>==
===Introduction===
===Introduction===
In the last lab session the team carried out a successful PCR reaction on the ''cotC-GFP-smtA'' BioBrick and cleaned up the products. The team also midi-prepped ''cotC-GFP-smtA'', ''kinA'', ''pGFP-rrnB'', ''pMUTIN4'' and ''pSB1AT3'' which will prove useful for other team members too. In addition, transformant ''E. coli'' cells containing the ''kinA'' BioBrick and the ''cotC-GFP-smtA'' BioBrick were grown up in 5ml LB cultures and then frozen down.
In the last lab session the team carried out a successful PCR reaction on the ''cotC-GFP-smtA'' BioBrick and cleaned up the products. The team also midi-prepped ''cotC-GFP-smtA'', ''kinA'', ''pGFP-rrnB'', ''pMUTIN4'' and ''pSB1AT3'' which will prove useful for other team members too. In addition, transformant ''E. coli'' cells containing the ''kinA'' BioBrick and the ''cotC-GFP-smtA'' BioBrick were grown up in 5ml LB cultures and then frozen down.
 +
However there were also things that we didn't accomplish. We had previously carried out a PCR reaction involving the two elements of our cadmium-sensitive logic AND gate - ''arsR'' and ''cadA''. But when the products were cleaned a large amount of ethanol remained in the sample and so when placed into agarose gel the sample dispersed. We then proceded to carry out a second attempt at the same PCR reaction.
However there were also things that we didn't accomplish. We had previously carried out a PCR reaction involving the two elements of our cadmium-sensitive logic AND gate - ''arsR'' and ''cadA''. But when the products were cleaned a large amount of ethanol remained in the sample and so when placed into agarose gel the sample dispersed. We then proceded to carry out a second attempt at the same PCR reaction.
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Today's lab session will continue straight on from the last lab session and fulfil the following objectives:
Today's lab session will continue straight on from the last lab session and fulfil the following objectives:
<br>
<br>
 +
[[Image:Team Newcastle 2009 iGEM 14-09-09 IMG 1136.JPG|200px|right]]
# Run products of PCR reaction involving the ''arsR'' BioBrick (''BBa_J33206'' in ''pSB1A2'' missing promoter) and ''cadA'' promoter region  
# Run products of PCR reaction involving the ''arsR'' BioBrick (''BBa_J33206'' in ''pSB1A2'' missing promoter) and ''cadA'' promoter region  
## If successful clean up product, run on agarose gel through electrophoresis, excise band and carry out gel extraction.  
## If successful clean up product, run on agarose gel through electrophoresis, excise band and carry out gel extraction.  
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===Procedure===
===Procedure===
-
====Cadmium-sensing logic AND gate====
+
====1) Cadmium-sensing logic AND gate====
[[Image:Team Newcastle 2009 iGEM Geldoc 2009-09-14 13hr 08min.jpg|400px|center]]
[[Image:Team Newcastle 2009 iGEM Geldoc 2009-09-14 13hr 08min.jpg|400px|center]]
<br>
<br>
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All of the DNA from the PCR reactions involving the ''cadA'' promoter region and the ''arsR'' BioBrick (''BBa_J33206'' without promoter) was inserted into agarose gel (in two separate wells of course) and DNA gel electrophoresis allowed to commence for 20 minutes under 80 volts.
All of the DNA from the PCR reactions involving the ''cadA'' promoter region and the ''arsR'' BioBrick (''BBa_J33206'' without promoter) was inserted into agarose gel (in two separate wells of course) and DNA gel electrophoresis allowed to commence for 20 minutes under 80 volts.
 +
[[Image:Team Newcastle 2009 iGEM 14-09-09 IMG 1128.JPG|200px|right]]
We also ran ''ara'' and ''pGFP-rrnB'' DNA on the gel (this is for the Stochastic Switch team).
We also ran ''ara'' and ''pGFP-rrnB'' DNA on the gel (this is for the Stochastic Switch team).
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|}
|}
<br>
<br>
 +
The two fragments then underwent a FastLigation reaction. This ligation only took 10 minutes. This is what we inserted into the ligation reaction:
The two fragments then underwent a FastLigation reaction. This ligation only took 10 minutes. This is what we inserted into the ligation reaction:
<br>
<br>
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'''Note: ''cadA'' promoter region DNA concentration was 17ng/ul; ''arsR'' BioBrick DNA concentration was 40ng/ul'''
'''Note: ''cadA'' promoter region DNA concentration was 17ng/ul; ''arsR'' BioBrick DNA concentration was 40ng/ul'''
-
====Ligating the ''cotC'' BioBrick with ''pMUTIN4''====
+
====2) Ligating the ''cotC'' BioBrick with ''pMUTIN4''====
Firstly the ''cotC'' BioBrick and the ''pMUTIN4'' DNA was cut with ''BamHI'' and ''HindIII'':
Firstly the ''cotC'' BioBrick and the ''pMUTIN4'' DNA was cut with ''BamHI'' and ''HindIII'':
<br>
<br>
{| border="1"
{| border="1"
|+ Contents of the ''cotC'' and ''pMUTIN4'' DNA digests
|+ Contents of the ''cotC'' and ''pMUTIN4'' DNA digests
-
!  !! ''cotC'' BioBrick !! ''pMUTIN4'' plasmid !!
+
!  !! ''cotC'' BioBrick (reaction 1) !! ''cotC BioBrick (reaction2) !! ''pMUTIN4'' plasmid !!
|-
|-
! DNA (ul)
! DNA (ul)
-
| 10 || 10 ||
+
| 30 || 10 || 20
|-
|-
! Restriction Buffer (ul)
! Restriction Buffer (ul)
-
| 2 || 2 ||
+
| 5 || 5 || 5
|-
|-
! ''BamHI'' Restriction Enzyme(ul)
! ''BamHI'' Restriction Enzyme(ul)
-
| 1 || 1 ||
+
| 1 || 1 || 1
|-
|-
! ''HindIII'' Restriction Enzyme(ul)
! ''HindIII'' Restriction Enzyme(ul)
-
| 1 || 1 ||
+
| 1 || 1 || 1
|-
|-
! Distilled water(ul)
! Distilled water(ul)
-
| 6 || 6 ||
+
| 13 || 33 || 23
|}
|}
<br>
<br>
 +
These digests were left to commence for 1 hour. Once this time had elapsed the two fragments of DNA were ligated together in the following way:
 +
<br>
 +
* 1ul of ''pMUTIN4''
 +
* 6ul of ''cotC''
 +
* 4ul of buffer solution
 +
* 1ul DNA ligase
 +
* 7ul sterile distilled water
 +
<br>
 +
==== 3) Transforming ''E. coli'' cells with two ligated products ====
 +
[[Image:Team Newcastle 2009 iGEM 14-09-09 IMG 1151.JPG|200px|right]]
 +
The two ligated products - the ''arsR''-''cadA'' promoter AND gate and the ''cotC-GFP-smtA'' BioBrick in ''pMUTIN4'' - were then transformed into ''DH5-alpha'' ''E. coli'' cells using [https://2009.igem.org/Team:Newcastle/Project/Labwork/PhilsProtocols#Transforming_DNA_.22Phil_Style.22 Phil's transformation by heat shock protocol]. There were only two changes to the protocol:
 +
<br>
 +
* For step 2 - the waiting time was 15 minutes instead of 30 minutes
 +
* For step 5 - the waiting time was also 15 minutes instead of 30 minutes.
 +
<br>
 +
All of the DNA from the ligation reactions was added to the competent cells. Once the ligations were completed, the cells were plated onto LB + antibiotic plates. The ''arsR''-''cadA'' promoter transformants were plated on LB + ampicillin plates whereas the ''cotC-GFP-smtA'' BioBrick in ''pMUTIN4'' transformant cells were plated on LB + chloramphenicol plates.
 +
 +
==<u>Sporulation Tuning/Chassis Team</u>==
 +
 +
===Introduction===
 +
 +
* Since we have got the colonys from sleB transformation, and we cultured 5ml overnight cells yesterday. Finally, we can carry out a Mini prep to check our result for transformation.
 +
* In case our transformation didn't work, we prepare digested sleB fragment as a backup.
 +
* For cwlJ Biobrick, we need to digest cwlJ PCR product and do another transformation.
 +
 +
<br>
 +
 +
===Experiment procedure===
 +
[[Image:Team Newcastle 2009 iGEM 14-09-09 IMG 1125.JPG|200px|right]]
 +
==== Mini Prep ====
 +
* We have cultured 10 colonies which can grow on the LB+Amp+Tet plate.
 +
* After overnight grow, we perform the Mini Prep to select the right colony which contains sleB clone.
 +
* The Mini Prep experiment followed the Phil's Mini Prep protocal
 +
<br>
 +
 +
==== Fast digest Mini prep result====
 +
* This digest is the simple test of our sleB transformation result, we use 20ul for the final volume.
 +
 +
  ddH2o                          7.0ul
 +
  10X fast digest buffer            2ul
 +
  Mini prep DNA                    10ul
 +
  Restriction enzyme 1            0.5ul
 +
  Restriction enzyme 2            0.5ul
 +
  -------------------------------------
 +
                          Total:  20ul 
 +
 +
* Here the restriction enzyme 1 and 2 were the same enzyme used for the digestion of sleB PCR result: EcoRI and speI.
 +
* 37C 1 hour. 
 +
* After one hour digestion, we loaded 10 digested samples in 0.8% agarose gel, 90v 30 - 45 mins.
 +
* Scanned the electrophoresis result in UV light.
 +
 +
==== Digest cwlJ and sleB PCR products ====
 +
<br>
 +
This digestion was carried out for the ligation and transformation.
 +
 +
* sleB digestion: sleB DNA has been cleaned up by PCR Clean up Kit after PCR.
 +
 +
  dd H2O                    7ul
 +
  10X fast digest buffer    7ul
 +
  Fast EcoRI                3ul
 +
  Fast speI                  3ul
 +
  sleB DNA                  50ul
 +
  ------------------------------
 +
                            70ul
 +
 +
* cwlJ digestion: cwlJ DNA has been cleaned up by PCR Clean up Kit after PCR.
 +
 +
  dd H2O                    7ul
 +
  10X fast digest buffer    7ul
 +
  Fast EcoRI                3ul
 +
  Fast speI                  3ul
 +
  sleB DNA                  50ul
 +
  ------------------------------
 +
                            70ul
 +
 +
==== Gel extraction of digested DNA ====
 +
[[Image:Team Newcastle 2009 iGEM 14-09-09 IMG 1145.JPG|200px|right]]
 +
* Pour the big well gel before digestion was finished.
 +
* After 1 hour digestion of cwlJ and sleB PCR products, we loaded the digested DNA in big well.
 +
* 90 volt, run for 30 - 45 mins.
 +
* Check the result under UV light.
 +
* Cut the right band and purified sleB and cwlJ DNA from agarose gel using Gel Extraction Kit(Sigma).
 +
 +
==== Midi culture colony which contains right sleB clone ====
 +
* Take out 200ul cells from Mini prep tube.(there should have 3.5ml cells left after Mini prep)
 +
 +
    Strain 1 was select here.
 +
 +
===Conclusion===
 +
* The electrophoresis result after digestion showed that 9 colonies had got the sleB clone successfully.
 +
 +
<br>
 +
[[Image:Team_newcastle_2009_igem_hjgel_140909_sleB.png‎ |400px|thumb|center|sleB Mini Prep result]]
 +
 +
===Futher plan===
 +
* Culture one strain for Midi Prep.
 +
* Ligation cwlJ and pSB1AT3 and do the transformation.
 +
* Ligation cwlJ with pSB1AT3:sleb to obtain a double clone.
 +
 +
{{:Team:Newcastle/Project/Labwork/CalTemplate}}
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{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Right}}
{{:Team:Newcastle/Right}}

Latest revision as of 12:16, 20 August 2010

Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG

Formal Lab Session - 14th September 2009

Team Newcastle 2009 iGEM 14-09-09 IMG 1131.JPG


Overview

Metal Sensor team

Introduction

In the last lab session the team carried out a successful PCR reaction on the cotC-GFP-smtA BioBrick and cleaned up the products. The team also midi-prepped cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3 which will prove useful for other team members too. In addition, transformant E. coli cells containing the kinA BioBrick and the cotC-GFP-smtA BioBrick were grown up in 5ml LB cultures and then frozen down.


However there were also things that we didn't accomplish. We had previously carried out a PCR reaction involving the two elements of our cadmium-sensitive logic AND gate - arsR and cadA. But when the products were cleaned a large amount of ethanol remained in the sample and so when placed into agarose gel the sample dispersed. We then proceded to carry out a second attempt at the same PCR reaction.

In today's practical we hope to ligate pMUTIN4 and the cotC-GFP-smtA BioBrick together - firstly cutting both fragments with BamHI and HindIII and then ligating the DNA together. We also hope to run the arsR and cadA promoter PCR products on agarose gel (in DNA gel electrophoresis) and if successful, clean up the sample. Then run all of the sample through agarose gel and excise the band produced, also extracting the gel from it afterwards. If all these events happen, we will also cut both the arsR and cadA fragments with BamHI and NheI and attempt overnight ligation.

Practical Outline

Today's lab session will continue straight on from the last lab session and fulfil the following objectives:

Team Newcastle 2009 iGEM 14-09-09 IMG 1136.JPG
  1. Run products of PCR reaction involving the arsR BioBrick (BBa_J33206 in pSB1A2 missing promoter) and cadA promoter region
    1. If successful clean up product, run on agarose gel through electrophoresis, excise band and carry out gel extraction.
    2. Cut cleaned and excised fragment with BamHI and NheI
    3. Carry out ligation (possibly an overnight ligation)
  2. Cut pMUTIN4 and cotC (i.e. PCR product 3) with BamHI and HindIII
    1. Ligate the fragments (possibly overnight)


Procedure

1) Cadmium-sensing logic AND gate

Team Newcastle 2009 iGEM Geldoc 2009-09-14 13hr 08min.jpg


The first step to take was to run the PCR products of reactions involving the arsR BioBrick (BBa_J33206 without it's original promoter) and the cadA promoter region through agarose gel in DNA gel electrophoresis. The initial views of the team was to carry out a test on the samples on some gel before excising the rest of the DNA when in gel. However we decided to place all of the DNA into gel and excise the band immediately, without conducting tests.

All of the DNA from the PCR reactions involving the cadA promoter region and the arsR BioBrick (BBa_J33206 without promoter) was inserted into agarose gel (in two separate wells of course) and DNA gel electrophoresis allowed to commence for 20 minutes under 80 volts.

Team Newcastle 2009 iGEM 14-09-09 IMG 1128.JPG

We also ran ara and pGFP-rrnB DNA on the gel (this is for the Stochastic Switch team).

The bands were then excised and cleaned using Sigma-Aldrich's GenElute Gel Extraction kit (along with the protocol). There was no changes to the protocol but a few important points to note:

  • The 'Spin Method' was the preferred route in the protocol to follow.
  • The mass of the gel bands were as follows:
    • arsR band = 0.285 grams
    • cadA promoter region band = 0.354 grams
    • ara band = 0.212 grams
    • pGFP-rrnB band = 0.233 grams
  • Therefore the amount of Gel Solublization Solution to be added to each of the bands were as follows:
    • arsR band = 860ul of solution to be added
    • cadA promoter region band = 1060ul of solution to be added
    • ara band = 640ul of solution to be added
    • pGFP-rrnB band = 700ul of solution to be added
  • Therefore the amount of isopropanol to be added to the DNA samples was as follows:
    • arsR band = 285ul of isopropanol to be added
    • cadA promoter region band = 354ul of isopropanol to be added
    • ara band = 212ul of isopropanol to be added
    • pGFP-rrnB band = 233ul of isopropanol to be added


Once the arsR (the BBa_J33206 BioBrick without promoter region) and cadA promoter DNA samples were cleaned, they were both cut using BamHI and NheI in the following way:

Contents of the arsR and cadA promoter digests
arsR BioBrick cadA promoter
DNA (ul) 10 10
Restriction Buffer (ul) 2 2
BamHI Restriction Enzyme(ul) 1 1
NheI Restriction Enzyme(ul) 1 1
Distilled water(ul) 6 6


The two fragments then underwent a FastLigation reaction. This ligation only took 10 minutes. This is what we inserted into the ligation reaction:

  • Linearised vector DNA = BBa_J33206 (arsR) without promoter - 1ul
  • Insert DNA = cadA promoter region - 7ul
  • 5x Rapid Ligation Buffer = 4ul
  • Water = 6ul
  • T4 DNA ligase = 1ul


Note: cadA promoter region DNA concentration was 17ng/ul; arsR BioBrick DNA concentration was 40ng/ul

2) Ligating the cotC BioBrick with pMUTIN4

Firstly the cotC BioBrick and the pMUTIN4 DNA was cut with BamHI and HindIII:

Contents of the cotC and pMUTIN4 DNA digests
cotC BioBrick (reaction 1) cotC BioBrick (reaction2) pMUTIN4 plasmid
DNA (ul) 30 10 20
Restriction Buffer (ul) 5 5 5
BamHI Restriction Enzyme(ul) 1 1 1
HindIII Restriction Enzyme(ul) 1 1 1
Distilled water(ul) 13 33 23


These digests were left to commence for 1 hour. Once this time had elapsed the two fragments of DNA were ligated together in the following way:

  • 1ul of pMUTIN4
  • 6ul of cotC
  • 4ul of buffer solution
  • 1ul DNA ligase
  • 7ul sterile distilled water


3) Transforming E. coli cells with two ligated products

Team Newcastle 2009 iGEM 14-09-09 IMG 1151.JPG

The two ligated products - the arsR-cadA promoter AND gate and the cotC-GFP-smtA BioBrick in pMUTIN4 - were then transformed into DH5-alpha E. coli cells using Phil's transformation by heat shock protocol. There were only two changes to the protocol:

  • For step 2 - the waiting time was 15 minutes instead of 30 minutes
  • For step 5 - the waiting time was also 15 minutes instead of 30 minutes.


All of the DNA from the ligation reactions was added to the competent cells. Once the ligations were completed, the cells were plated onto LB + antibiotic plates. The arsR-cadA promoter transformants were plated on LB + ampicillin plates whereas the cotC-GFP-smtA BioBrick in pMUTIN4 transformant cells were plated on LB + chloramphenicol plates.

Sporulation Tuning/Chassis Team

Introduction

  • Since we have got the colonys from sleB transformation, and we cultured 5ml overnight cells yesterday. Finally, we can carry out a Mini prep to check our result for transformation.
  • In case our transformation didn't work, we prepare digested sleB fragment as a backup.
  • For cwlJ Biobrick, we need to digest cwlJ PCR product and do another transformation.


Experiment procedure

Team Newcastle 2009 iGEM 14-09-09 IMG 1125.JPG

Mini Prep

  • We have cultured 10 colonies which can grow on the LB+Amp+Tet plate.
  • After overnight grow, we perform the Mini Prep to select the right colony which contains sleB clone.
  • The Mini Prep experiment followed the Phil's Mini Prep protocal


Fast digest Mini prep result

  • This digest is the simple test of our sleB transformation result, we use 20ul for the final volume. 
 ddH2o                           7.0ul
 10X fast digest buffer            2ul
 Mini prep DNA                    10ul
 Restriction enzyme 1            0.5ul
 Restriction enzyme 2            0.5ul
 -------------------------------------
                         Total:   20ul
  • Here the restriction enzyme 1 and 2 were the same enzyme used for the digestion of sleB PCR result: EcoRI and speI.
  • 37C 1 hour.
  • After one hour digestion, we loaded 10 digested samples in 0.8% agarose gel, 90v 30 - 45 mins.
  • Scanned the electrophoresis result in UV light.

Digest cwlJ and sleB PCR products


This digestion was carried out for the ligation and transformation.

  • sleB digestion: sleB DNA has been cleaned up by PCR Clean up Kit after PCR. 
 dd H2O                     7ul
 10X fast digest buffer     7ul
 Fast EcoRI                 3ul
 Fast speI                  3ul
 sleB DNA                  50ul
 ------------------------------
                           70ul
  • cwlJ digestion: cwlJ DNA has been cleaned up by PCR Clean up Kit after PCR. 
 dd H2O                     7ul
 10X fast digest buffer     7ul
 Fast EcoRI                 3ul
 Fast speI                  3ul
 sleB DNA                  50ul
 ------------------------------
                           70ul

Gel extraction of digested DNA

Team Newcastle 2009 iGEM 14-09-09 IMG 1145.JPG
  • Pour the big well gel before digestion was finished.
  • After 1 hour digestion of cwlJ and sleB PCR products, we loaded the digested DNA in big well.
  • 90 volt, run for 30 - 45 mins.
  • Check the result under UV light.
  • Cut the right band and purified sleB and cwlJ DNA from agarose gel using Gel Extraction Kit(Sigma).

Midi culture colony which contains right sleB clone

  • Take out 200ul cells from Mini prep tube.(there should have 3.5ml cells left after Mini prep) 
   Strain 1 was select here.

Conclusion

  • The electrophoresis result after digestion showed that 9 colonies had got the sleB clone successfully.


sleB Mini Prep result

Futher plan

  • Culture one strain for Midi Prep.
  • Ligation cwlJ and pSB1AT3 and do the transformation.
  • Ligation cwlJ with pSB1AT3:sleb to obtain a double clone.
July
MTWTFSS
    [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_July_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_July_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_July_2009&action=edit 5]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_July_2009&action=edit 12]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_July_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/14_July_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_July_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_July_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_July_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_July_2009&action=edit 19]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_July_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_July_2009&action=edit 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_July_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_July_2009 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_July_2009&action=edit 26]
[http://2009.igem.org/Team:Newcastle/Labwork/27_July_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_July_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_July_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_July_2009 30] [http://2009.igem.org/Team:Newcastle/Labwork/31_July_2009 31]
August
MTWTFSS
          [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_August_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_August_2009&action=edit 2]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_August_2009&action=edit 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_August_2009 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_August_2009 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_August_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_August_2009 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_August_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_August_2009&action=edit 9]
[http://2009.igem.org/Team:Newcastle/Labwork/10_August_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_August_2009 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_August_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_August_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_August_2009 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_August_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_August_2009&action=edit 16]
[http://2009.igem.org/Team:Newcastle/Labwork/17_August_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_August_2009 18] [http://2009.igem.org/Team:Newcastle/Labwork/19_August_2009 19] [http://2009.igem.org/Team:Newcastle/Labwork/20_August_2009 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_August_2009 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_August_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_August_2009&action=edit 23]
[http://2009.igem.org/Team:Newcastle/Labwork/24_August_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_August_2009 25] [http://2009.igem.org/Team:Newcastle/Labwork/26_August_2009 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_August_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_August_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_August_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_August_2009&action=edit 30]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_August_2009&action=edit 31]
September
MTWTFSS
  [http://2009.igem.org/Team:Newcastle/Labwork/1_September_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_September_2009 2] [http://2009.igem.org/Team:Newcastle/Labwork/3_September_2009 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_September_2009 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_September_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_September_2009&action=edit 6]
[http://2009.igem.org/Team:Newcastle/Labwork/7_September_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_September_2009 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_September_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_September_2009 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_September_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_September_2009&action=edit 13]
[http://2009.igem.org/Team:Newcastle/Labwork/14_September_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_September_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_September_2009 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_September_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_September_2009 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_September_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_September_2009&action=edit 20]
[http://2009.igem.org/Team:Newcastle/Labwork/21_September_2009 21] [http://2009.igem.org/Team:Newcastle/Labwork/22_September_2009 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_September_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_September_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_September_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_September_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_September_2009&action=edit 27]
[http://2009.igem.org/Team:Newcastle/Labwork/28_September_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_September_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_September_2009 30]
October
MTWTFSS
      [http://2009.igem.org/Team:Newcastle/Labwork/1_October_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_October_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_October_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_October_2009&action=edit 4]
[http://2009.igem.org/Team:Newcastle/Labwork/5_October_2009 5] [http://2009.igem.org/Team:Newcastle/Labwork/6_October_2009 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_October_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_October_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_October_2009 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_October_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_October_2009&action=edit 11]
[http://2009.igem.org/Team:Newcastle/Labwork/12_October_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_October_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_October_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_October_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_October_2009 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_October_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_October_2009&action=edit 18]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_October_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_October_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_October_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_October_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_October_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/24_October_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_October_2009&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_October_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_October_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/28_October_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_October_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_October_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_October_2009&action=edit 31]



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