Team:Newcastle/Labwork/11 September 2009

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The following procedures were carried out:
The following procedures were carried out:
====Freeze the ''kinA'' and ''cotC-GFP-smtA'' transformant cells====
====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 [https://2009.igem.org/Team:Newcastle/Project/Labwork/PhilsProtocols#Freezing_Strains_into_the_TPA_Collection protocol]
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====PCR the ''cotC-GFP-smtA'' BioBrick with ''pMK15'' and ''pMK16''====
====PCR the ''cotC-GFP-smtA'' BioBrick with ''pMK15'' and ''pMK16''====
====Midi-prep ''cotC-GFP-smtA'', ''kinA'', ''pGFP-rrnB'', ''pMUTIN4'' and ''pSB1AT3''====
====Midi-prep ''cotC-GFP-smtA'', ''kinA'', ''pGFP-rrnB'', ''pMUTIN4'' and ''pSB1AT3''====

Revision as of 00:16, 14 September 2009

Lab Work - 11/09/09

Metal Sensing Team

Introduction and Outline

So far we have transformed DH5-alpha E. coli cells with two of our synthesized BioBricks, which are the cotC-GFP-smtA and the kinA BioBricks. 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



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