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Team:Newcastle/Labwork/11 September 2009
From 2009.igem.org
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==<u>Metal Sensing Team</u>== | ==<u>Metal Sensing Team</u>== | ||
===Introduction and Outline=== | ===Introduction and Outline=== | ||
| - | So far we have transformed ''DH5-alpha E. coli'' cells with two | + | So far we have transformed ''DH5-alpha E. coli'' cells with two synthesized BioBricks, which are the ''cotC-GFP-smtA'' and the ''kinA'' BioBricks. The ''cotC-GFP-smtA'' BioBrick is involved within the metal sensing sub-project whereas the ''kinA'' BioBrick is involved with the Stochastic Switch sub-project. The Metal Sensing team have since taken these transformant colonies and used them to set up LB+kanamycin cultures for mini-preps and midi-preps; these were then left overnight in the orbital shaking incubator and are awaiting processing today. However there are a range of tasks which need to be accomplished today; these are listed below: |
<br> | <br> | ||
* '''Prepare Mini-preps of the ''kinA'' and ''cotC-GFP-smtA'' transformants''' | * '''Prepare Mini-preps of the ''kinA'' and ''cotC-GFP-smtA'' transformants''' | ||
Revision as of 09:26, 15 September 2009
Project
Sub-projects
Wet Lab
Meetings
Planning
Links
Lab Work - 11/09/09
Metal Sensing Team
Introduction and Outline
So far we have transformed DH5-alpha E. coli cells with two synthesized BioBricks, which are the cotC-GFP-smtA and the kinA BioBricks. The cotC-GFP-smtA BioBrick is involved within the metal sensing sub-project whereas the kinA BioBrick is involved with the Stochastic Switch sub-project. The Metal Sensing team have since taken these transformant colonies and used them to set up LB+kanamycin cultures for mini-preps and midi-preps; these were then left overnight in the orbital shaking incubator and are awaiting processing today. However there are a range of tasks which need to be accomplished today; these are listed below:
- Prepare Mini-preps of the kinA and cotC-GFP-smtA transformants
- Carry out the mini-prep processes
- Analyse the mini-prep DNA by treating with restriction enzymes.
- PCR the cotC-GFP-smtA BioBrick with pMK15 and pMK16
- Set up the PCR
- Run the PCR for the correct duration
- Analyse PCR products through DNA gel electrophoresis
- Clean up PCR products if PCR reaction proves to be successful
- Midi-prep cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3
- Carry out the five midi-preps.
- Quantify the midi-prep samples
- Run the midi-prep samples on agarose gel through DNA gel electrophoresis
- Run products from a PCR reaction involving arsR and cadA DNA
- Run PCR products on gel
- If successful, clean up fragments, run on gel again, cut with restriction enzymes NheI and BamHI, clean and then ligate together fragments.
Procedure
The following procedures were carried out:
Freeze the kinA and cotC-GFP-smtA transformant cells
Instead of preparing mini-prep samples from the two groups of transformant E.coli cells (i.e. kinA transformed E.coli cells and cotC-GFP-smtA transformed cells), we instead took the decision to freeze the cells and analyse the midi-prep samples - this will both save time and also provide a back-up stock of cells for future work. This was done using the protocol
PCR the cotC-GFP-smtA BioBrick with pMK15 and pMK16
Midi-prep cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3
Run products from a PCR reaction involving arsR and cadA DNA
Conclusions
News
Events
- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
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]
