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use of pGEM -tT easy vectors


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#1 Debo

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Posted 06 July 2011 - 08:26 PM

I have been trying to ligate my product into T easy vector . but the clones don seem to come at all. tried changing competent cells . its still not working .
my PCR products have been made by Pfu polymerase which has high fidelity and does not add A in the end therefore i did A tailing for them . what could be the prob ?

Edited by Debo, 06 July 2011 - 08:26 PM.


#2 Adrian K

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Posted 07 July 2011 - 07:43 AM

Just try once: use normal Taq, do the PCR again, and try ligate again into your vector. If this succeed, this means you might did sthg wrong during the A-tailing procedure. If not, it could be your vector/ligation problem.

Just 2 cents.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 Adrian K

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Posted 07 July 2011 - 07:44 AM

Just try once: use normal Taq, do the PCR again, and try ligate again into your vector. If this succeed, this means you might did sthg wrong during the A-tailing procedure. If not, it could be your vector/ligation problem.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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