2nd trial of colony-PCR - (Oct/14/2006 )
to my JOY, all the 30 colonies picked for 2 sets of PCR this morning yielded the expected PCR products. The PCR results suggested the presence of the 1st insert, 3rd insert and the Erm resistance suggested the presence of 2nd insert, the ErmR cassette! It seems that the colonies carry the desired recombinant plasmid. And of course, I will pick about 5-6 for miniprep and double check the RE pattern.
And indeed, the failure of PCR yesterday was due to the template suspension.....The clearer cell suspension used this morning gave positive results in each sample picked.
thanks guys for all your tips and comments along this ligation and screening process!
Plasmid problem of the week solved.
I checked the restriction profile (of both single digestion and double-digestion) today--all 6 plasmids tested seemed to be correct...Tomorrow I will send 2 plasmids off for sequencing, although I am now pretty happy and confident with the 4-way ligation result.
The next challenge would be to conjugate it to my Vibrio wildtype strain and select for clean mutant which picks ErmR casettee while loses the target gene (to be deleted) via two events of recombination....
Indeed, thanks so much for all the tips and suggestions you guys have contributed!