lambda PCR

Shiao Y. Wang sywang at whale.st.usm.edu
Mon Apr 18 22:09:51 EST 1994


Przemko (przemko at reks.uia.ac.be) wrote:
: We are using PCR directly on bacterial colonies to get inserts
: (for cloning or to prepare probe or to check cloning) and it works
: like a dream. No purification, no nothing. Just pick a colony and go.
: Now we have to do the same but on lambda plaques. It appears however
: that it is not so easy. We get a lot of background, nonspecific bands etc.
: We use commercial primers (Promega, I believe).

We do PCR on lambda clones routinely. We remove a plug of the plaque and
soak it TE or phage dilution buffer for 1 hr and use 1 ul of the liquid
for PCR. Couldn't be easier (like your experience w/ colonies). If you are
having background problems, I suspect it's either due to non-specificity
of the primers or low annealing temperature. You mentioned that you used
commercial primers, were these established primers like gt10 forward and
reverse primers? If so they should work. I would double check and make
sure you're using the right primers. Sounds simple but we all make simple
mistakes.



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