Buffer conditions for Pfu? - (Jul/30/2005 )
Hey,
´
I try to clone ~ 1000 bp fragment with TOPO TA cloning kit from invitrogen.
Therefore I do PCR using Taq, clone it into the vector and unfortunatley get mutations in my sequence of interest! I have no idea why Taq starts to annoy me but it´s becoming a pain in the neck, so I thought about using Pfu polymerase and afterwards Taq for the 3´A overhangs.
I found an (probably) old vial of Pfu but do not have any special buffers, so I think I will have to try Taq buffer (invitrogen) for my Pfu (stratgene).
Does anybody here ever mixed these ingredients? Do I have to consider special things about using "a-non-optimized" buffer?
Thanks in advance for your thoughts.
Cheers
Hi,
I am using NEB Taq and mix it with Biozym Pfu (NEB Buffer). The Pfu is expired 2001....Ive cloned an Fragmet of 1500 bp with it and from 3 Preps just one had a single base-exchange...so I think its ok.....but nobody would garantee for it.......but when I used HighFi-Taq from Roche there were also some base exchanges...
Greetz
K_R
´
I try to clone ~ 1000 bp fragment with TOPO TA cloning kit from invitrogen.
Therefore I do PCR using Taq, clone it into the vector and unfortunatley get mutations in my sequence of interest! I have no idea why Taq starts to annoy me but it´s becoming a pain in the neck, so I thought about using Pfu polymerase and afterwards Taq for the 3´A overhangs.
I found an (probably) old vial of Pfu but do not have any special buffers, so I think I will have to try Taq buffer (invitrogen) for my Pfu (stratgene).
Does anybody here ever mixed these ingredients? Do I have to consider special things about using "a-non-optimized" buffer?
Thanks in advance for your thoughts.
Cheers
I often use Pfu from Promega,and it is attached with Pfu buffer,and I found it is inefficient if the target fragment is beyond 1000bp.best wishes!
´
I try to clone ~ 1000 bp fragment with TOPO TA cloning kit from invitrogen.
Therefore I do PCR using Taq, clone it into the vector and unfortunatley get mutations in my sequence of interest! I have no idea why Taq starts to annoy me but it´s becoming a pain in the neck, so I thought about using Pfu polymerase and afterwards Taq for the 3´A overhangs.
I found an (probably) old vial of Pfu but do not have any special buffers, so I think I will have to try Taq buffer (invitrogen) for my Pfu (stratgene).
Does anybody here ever mixed these ingredients? Do I have to consider special things about using "a-non-optimized" buffer?
Thanks in advance for your thoughts.
Cheers
I often use Pfu from Promega,and it is attached with Pfu buffer,and I found it is inefficient if the target fragment is beyond 1000bp.best wishes!
Hi
As I know Pfu requires MgSo4 whereas Taq requires MgCl2.So their buffers contain different salts I think. May be it helps...