Team:Newcastle/Labwork/16 September 2009

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Formal Lab Session - 16th September 2009

Stochastic Switch Team

Today we ran the ara PCRs from yesterday on a gel and got a really good result, this product was then cleaned, cut with EcoRI and SpeI, cleaned again, then ligated with the pre-cut pSB1AT3 plasmid backbone.

Today we also did minipreps of 12 each of the sac and ara cultures... however unfortunately today the centrifuge we had been relying on broke, and therefore all three teams were forced to use one small centrifuge. This meant that the minipreps could end up being of poor quality due to waiting times for the centrifuge.


Metal Sensing Team

Introduction

In the last lab session, the Metal Sensing picked 12 colonies from a plate containing E. coli + cotC-GFP-smtA transformants and used them to inoculate 12 tubes containing LB + ampicillin - these wil be used for mini-preps today. In yesterday's session we also discovered that the attempts to transform E. coli with the arsR-cadA cadmium sensor failed, so on that same day we carried out a second attempt at transforming E. coli with this BioBrick.

Today's lab session will be focussed on carrying out the mini-preps for the 12 colonies of potential E. coli + cotC-GFP-smtA transformants and based on analysis, will determine whether midi-preps will follow or whether we need to reattempt another transformation (we would, of course, have to carry out another ligation reaction of the cotC BioBrick and the pMUTIN4 plasmid first)

To prepare for the eventuality that the ligations, transformations and midi-preps of the cotC-GFP-smtA in pMUTIN4 worked, the "Evening before transformations day" steps of the Bacillus subtilis transformation protocol will be carried out too.

The plate containing the cadmium-sensor E. coli transformants will also be studied - if there are colonies, 12 of them will be prepared for mini-preps; if there is no success then ligation of the [http://partsregistry.org/Part:BBa_J33206 BBa_J33206 BioBrick] (promoter removed) with the cadA promoter will be carried out - E. coli cells will then be transformed with this.

Practical Outline

  1. Carry out mini-preps on the 12 colonies of potential E. coli + cotC-GFP-smtA (in pMUTIN4) transformants
  2. Digest these mini-preps with BamHI and HindIII'
  3. Analyse the mini-prep digests by DNA gel electrophoresis
    1. If there are any successes, prepare midi-prep cultures from leftover overnight incubations
    2. If no colonies present the desired DNA then conduct another ligation of cotC with pMUTIN4 and transform E. coli with this ligated product.
  4. Carry out the "Evening before trasnformations" step in the Bacillus subtilis transformation protocol.

Procedure


July
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    [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
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[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
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[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]



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