IGEM:IMPERIAL/2006/LabCalendar/2006-8-10
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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:
- Enzymes: HindIII, NdeI (Red buffer) - Expected length of DNA fragments: 1856bp and 2836bp
- Enzymes: BglII, Pvu II (Green buffer) - Expected length of DNA fragments: 708 bp and 3984bp
- 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:
- Expected length of DNA fragments: 3615bp
- Expected length of DNA fragments: 3615bp
- Expected length of DNA fragments: 3615bp
Vector plasmid: 6B:
- Expected length of DNA fragments: 3140bp
- Expected length of DNA fragments: 3140bp
- 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
