wrong sequencing of T clones - (May/24/2007 )
Hi all,
I am a grrenhand in molecular biology. Recently, I am doing the molecular cloning. I amplified my target fragment from the expression plasmid with the Ex-Taq polymerase (TaKaRa corporation), and purified my PCR products with gel-extraction. Then ligased into the T vector. T clones were sequenced. But there were several mutation in the target sequences. I am puzzled with the results, as the Ex-Taq polymerase has the 3'-5' exonulease activity. Why my sequences contain so many mutations (upto 5 mutations)? Could anyone help me? How do you get the exact sequence? Whether should I get some more T clones for re-sequencing ?
Thanks in advance.
I am a grrenhand in molecular biology. Recently, I am doing the molecular cloning. I amplified my target fragment from the expression plasmid with the Ex-Taq polymerase (TaKaRa corporation), and purified my PCR products with gel-extraction. Then ligased into the T vector. T clones were sequenced. But there were several mutation in the target sequences. I am puzzled with the results, as the Ex-Taq polymerase has the 3'-5' exonulease activity. Why my sequences contain so many mutations (upto 5 mutations)? Could anyone help me? How do you get the exact sequence? Whether should I get some more T clones for re-sequencing ?
Thanks in advance.
First thing to do is to check the chromotography reads to make sure the "mutations" are real. Could the "mutations" be an artifact of a poor sequence read? I
Next, did you sequence from both direction, forward and reverse? Sequencing both direction, means that any mutations would appear in both reads... confirming it validity.
I would definately seqence more clones (my minimum for something important is 3) from both the forward and reverse direction.
Now on to another aspect, how long is the DNA segment that was clone? Is it unsually rich in mononucleotide repeats?
First thing to do is to check the chromotography reads to make sure the "mutations" are real. Could the "mutations" be an artifact of a poor sequence read?
Next, did you sequence from both direction, forward and reverse? Sequencing both direction, means that any mutations would appear in both reads... confirming it validity.
I would definately seqence more clones (my minimum for something important is 3) from both the forward and reverse direction.
Now on to another aspect, how long is the DNA segment that was clone? Is it unsually rich in mononucleotide repeats?
Thank you very much.
The sequence is about 1500bp, and it is not rich in mononucleotide repeats.
I sequenced it from both direction. The mutations are upto 5, I think they are not artifacts.
I have definate more clones for sequencing.
Thanks again for your constructive advice.
I am a grrenhand in molecular biology. Recently, I am doing the molecular cloning. I amplified my target fragment from the expression plasmid with the Ex-Taq polymerase (TaKaRa corporation), and purified my PCR products with gel-extraction. Then ligased into the T vector. T clones were sequenced. But there were several mutation in the target sequences. I am puzzled with the results, as the Ex-Taq polymerase has the 3'-5' exonulease activity. Why my sequences contain so many mutations (upto 5 mutations)? Could anyone help me? How do you get the exact sequence? Whether should I get some more T clones for re-sequencing ?
Thanks in advance.
Hello,
How many cycles did u run your PCR?
I think you can try pfu (promega) in your PCR. This is a high fidelity enzyme. I used it to clone a 3.6kb gene and I ran 30 cycles only.
Hope this can help.