Team:Newcastle/Labwork/14 September 2009

From 2009.igem.org

(Difference between revisions)
(Formal Lab Session - 14th September 2009)
Line 123: Line 123:
<br>
<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.
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.
 +
 +
<center>
 +
{|cellpadding="10"
 +
|{{#calendar: title=Team:Newcastle/Labwork |year=2009 | month=07 }}
 +
|{{#calendar: title=Team:Newcastle/Labwork |year=2009 | month=08 }}
 +
|{{#calendar: title=Team:Newcastle/Labwork |year=2009 | month=09 }}
 +
|}
 +
</center>
{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Right}}
{{:Team:Newcastle/Right}}

Revision as of 15:57, 10 October 2009


Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG

Formal Lab Session - 14th September 2009

Metal Sensing 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:

  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.

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

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.

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]



News

Events

Social Net

  • Newcastle iGEM Twitter
  • [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
  • [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]