Hello everyone , recently i'll trying amplify by PCR a fragment of 5 kb .... anyone knows articles talking about that ... .without use long PCR kit? is posible?
thanks!
but i can`t make it because the gen worked is highly similar to another and the smaller fragment for see a specific amplification is 5kb .... for that reason i am searching literature who worked with big fragments. But i gonna search the phusion polimerase ..:) thanks
You can try TaKaRa LA Taq for long PCR products and also GC Rich sequences. Good luck!
Agree with Luis. Takara LA Taq works for us. You need to be sure to follow the primer and dNTP concentrations listed in the LA taq protocol. Long fragments need more dNTP and less primer.
Hi,
you can use Vent exo- polymerase from NEB for the amplification of large DNA fragments... other wise split the gene into 2.5kb and 2.5kb and prepare one forword and one reverse and the two overlapping central primers so that u can cover total 5kb gene...
Think of using high fidelity polymerase such as Pfu or Vent polymerase
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