lambda PCR

chai_z at wehi.edu.au chai_z at wehi.edu.au
Wed Apr 20 19:26:49 EST 1994


In article <2ovi1v$jke at server.st.usm.edu>, sywang at whale.st.usm.edu (Shiao Y. Wang) writes:
> 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

I pick a tiny piece of the agar within the plaque, using either a fine end 
tooth stick or pippet tip end and put it into the 10-20ul PCR reaction,
overlany the mineral oil and then run the PCR. It worked beautifully all the 
time in my hand.

I used lambda gt10 forward and reverse primers, annealing temp. 50-55 deg. C.
with taq or Tth enzyme.

Zhonglin Chai




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