Colony PCR of Mouse SMC1fs scFv

2015-01-13

azim58 - Colony PCR of Mouse SMC1fs scFv

The initial attempt of the colony PCR using the dynazyme protocol yielded
results with far too many bands. Andrey thinks that there may have been
too much genomic DNA relative to the plasmid DNA and the primers, and he
suggests that we perform an overnight culture colony PCR. With an
overnight culture PCR, one does not have to keep track of the numbering
of the colonies on the plate, the band results on the gel are cleaner,
and then one can do the miniprep and sequencing straight from the culture
right after seeing the results of the gel.

Andrey said that I might also want to check the melting temperatures of
the primers.

* Primers to check for scFv

Plan

* Make overnight culture of colonies
* Perform 3 sets of PCR for comparison
  o regular colony PCR with dynazyme
  o regular colony PCR with Advantage
  o overnight culture colony PCR with dynazyme
  o overnight culture colony PCR with Advantage

Advantage PCR of miniprep for SMC1fs scFv

We were not able to detect any appropriately sized bands at the expected
size (750 bp).
I have double checked that we are using the right primers. We are as can
be seen here:
https://azim58.wikispaces.com/file/view/4+PCR+Protocol+to+Amplify+Linked+He
avy+and+Light+Chains+101110.doc

I have double checked that we really should be expecting a 750 bp band.
We should since this is what I obtained when I amplified the scFv
fragments after constructing them.
https://azim58.wikispaces.com/Amplified+Antibody+Genes+Results+Summary

There are two things I think I could try from here. I could try to cut
out the fragment I expect to see. I could also try to do a PCR in which I
add additional primers, enzymes, after several cycles have been run and
then run a few more cycles with these additional components. I could also
try to do a PCR which is run exactly the same way that I ran the PCR
before (with iProof and with 30 cycles and everything). Actually before I
think I did 20 cycles, added extra primer and polymerase, and then did 10
more cycles.

I think I'll try the restriction digest first.

Restriction Digest SfiI and NotI 090611.xls

The restriction digest revealed that I don't seem to have an insert.

I think there are probably some inserts in the whole library, but there
are probably just too many self-ligated plasmids. Maybe this could be
prevented by running the gel longer to separate cut from uncut plasmid. I
ran the gel of the cut plasmid before the ligation on 8-25-11.

In order to troubleshoot these problems I will do two things.

Cut plasmids with BamH1 and HindIII 090711
Cut and ligate rCompTT with PCANTAB5E 090711

"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-18-2014d1351\wikispaces download 01-18-2014d1351\01-18-2014d1352\Cut_SMC1fs_scFv_Plasmids_2.jpg"

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