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ligation - DNA present when run on gel, absent when using PCR!

ligation cloning colony pcr

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#1 fungirl 10

fungirl 10


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Posted 26 January 2012 - 01:53 PM

I am trying to insert 4kb into pGEM T-easy vector. I used Phusion polymerase, PCR purified, and A-tailed product. Went on with ligation. Ran ligation mixture on gel to visualize DNA and expected size bands were present. Then ran PCR on the ligation mixture using M13 primers (suggested to me by PI) and I have no bands. Transformed and have yet to find a positive clone using PCR. Screening with M13 primers. Should I use my sequence specific primers? Or use in in the vector and in the insert? I don't want to do this as I will have to set two reactions per sample to test directionality.

Then I tried inserting a different gene that was 2kb. I performed the same procedures and was able to see my DNA by running the ligation mixture on the gel AND by PCR. Then transformed. Used colony PCR to test six colonies and five out of six had the insert. So should I give up searching for my experimental insert since it did not amplify using PCR? And is colony pcr the best way to screen many colonies quickly?

Any advice is appreciated.

Edited by fungirl 10, 26 January 2012 - 01:54 PM.

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