What's the longest overhanging primer seq. you have used? - (Jun/18/2010 )
I want to include loxP sites flanking a gene that I want to amplify and then clone it into an expression vector. This is around 34 bp (+ 6 bp for a restriction site) that I must tag on to my primers. Any feedback will be appreciated.
should be absolutely no problem ...use 50bp overhangs without any problems regularly.
6 mer is quite ok, I've gone up to 8 for overhangs.
A friend of mine used 42 mer primer last time and she had no problem.
in case your PCR product digestion doesn't work, first clone the PCR product into a TA vector, or CloneJet vectors from Fermentas, then digest and clone into the second vector. This way you'll end up with many more colonies.
snpsnooper on Jun 18 2010, 09:16 AM said: