Team:Newcastle/Labwork/14 September 2009

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==Metal Sensing team==
==Metal Sensing team==
===Introduction===
===Introduction===
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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.
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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 need to attempt this PCR again and run the resulting fragments through gel.
===Practical Outline===
===Practical Outline===

Revision as of 21:44, 5 October 2009

Lab Work - 14/09/09

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 need to attempt this PCR again and run the resulting fragments through gel.

Practical Outline

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

  • 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.
    • Cut cleaned and excised fragment with BamHI and NheI
    • Carry out ligation (possibly an overnight ligation)
  • Cut pMUTIN4 and cotC (i.e. PCR product 3) with BamHI and HindIII
    • Ligate the fragments (possibly overnight)


Procedure



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