amplification linear ligation
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Question raised on 2010-12-11 20:29
Hi everybody,
I have a linear ligation product composed of 2 pieces (about 3kb in size). When I ran the ligation reaction on the gel, there were some unspecific bands as well as a faint band of around 3kb which seems to be the ligated band of interest. So I tried to amplify the 3kb band using Forward primer of the 1st fragment and Reverse primer of the 2nd one. Unfortunately I get only smear after amplification. Even changing annealing temperature (from 60-70) didn't affect positively.
I use high fidelity Phusion polymerase which recommend higher Ta (Tm+3), however it doesn't work. Also I tried different Mg concentration, but without promising results.
Tm(F)= 68.8
Tm®= 67.3
the questions are;
1. How can I ligate two or more linear fragments much more effectively and with higher yield for band of interest?
2. How can I amplify the ligated band of interest which is faint?
Should it seem possible to you kindly help me to find the solution.
many thanks
I have a linear ligation product composed of 2 pieces (
I use high fidelity Phusion polymerase which recommend higher Ta (Tm+3), however it doesn't work. Also I tried different Mg concentration, but without promising results.
Tm(F)= 68.8
Tm®= 67.3
the questions are;
1. How can I ligate two or more linear fragments much more effectively and with higher yield for band of interest?
2. How can I amplify the ligated band of interest which is faint?
Should it seem possible to you kindly help me to find the solution.
many thanks
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