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.
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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 ?
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 ?
#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.
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
..."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
..."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
