IGEM:IMPERIAL/2006/LabCalendar/2006-8-10

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Contents

Immunotag PCR

  • Ran gel of PCR products
  • However nothing showed up and our problems with PCR continue
  • Dr Mann is running both the lox and immunotag PCRs in his lab.
  • Tomorrow we find out whether we are in actual fact incompetent or there is something genuinely wrong with our protocol/primers.

Miniprep and digest

  • 12 colonies from ligation 12D -> 2H were minipreped and digested with EcoR1 and Pst1
  • Update...running gels do not seem to show any successful ligation. Perhaps we should try regrowing 12D and 2H and maxiprep again?

Control Digest for finalized prey cell J37015 & pSB1A2

Three different digests that cut either the part twice or the part and plasmid were set up to assess whether ligations were correctly made:

  1. Enzymes: HindIII, NdeI (Red buffer) - Expected length of DNA fragments: 1856bp and 2836bp
  2. Enzymes: BglII, Pvu II (Green buffer) - Expected length of DNA fragments: 708 bp and 3984bp
  3. Enzymes: BglII, Not II (Organge buffer - Expected length of DNA fragments: 899bp and 1738bp and 2055bp

As control, the insert plasmid and the vector plasmid are digested in addition to the recombinant plasmid above. Insert plasmid: 12D->24A:

  1. Expected length of DNA fragments: 3615bp
  2. Expected length of DNA fragments: 3615bp
  3. Expected length of DNA fragments: 3615bp

Vector plasmid: 6B:

  1. Expected length of DNA fragments: 3140bp
  2. Expected length of DNA fragments: 3140bp
  3. Expected length of DNA fragments: 2055bp and 1085bp
  • Left digests in the fridge overnight since it was too late to run the gel. The gel will be run tomorrow morning.

Culture grown up

  • T9002 and J37015 grown up and left overnight for possible testing tomorrow

Electroporation

  • Electroporated the ligations from yesterday. Need to culture them tomorrow then miniprep


Cre PCR

  • PCR has been set up to run overnight
  • The aim is to amplify out the Cre sequence from the plasmid that we have (it has no usefully placed restriction sites)
  • Gel to be run tomorrow to see if it is successful
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