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

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: As control, the insert plasmid and the vector plasmid are digested in addition to the recombinant plasmid above. Insert plasmid: 12D->24A: Vector plasmid: 6B:
 * 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
 * 1) Expected length of DNA fragments: 3615bp
 * 2) Expected length of DNA fragments: 3615bp
 * 3) Expected length of DNA fragments: 3615bp
 * 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