Taq vs. T4 dna polymerse: difference? - (Jun/10/2008 )
What is the difference in application btw these two enzymes? Would it matter to use either of them in cloning? One produces dAs at the end the other one creates blunt ends. Anybody has an idea if it might make a difference in consequent digestion with RE?
T4 is a bacteriophage of E. coli. It functions as a 5' -> 3' DNA polymerase and a 3' -> 5' exonuclease, but does not have 5' -> 3' exonuclease activity. This is same as klenow fragment of DNA polymerase 1.(if u remember)
In general, T4 DNA polymerase is used for the same types of reactions as Klenow fragment, particularly in blunting the ends of DNA with 5' or 3' overhangs. Mainly used for blunt end ligations.If you go to the NEB website, you will find info about this.
But, taq Polymerase has-- 1) 5'-3' polymerase activity
2)has 5' to 3' exonuclease activity
3) No 3' - 5' proof reading activity
4) and ideal for T-A cloning
In my opinion, it matters a lot if u use them for cloning. and yes it makes a difference for the RE analysis also.
If anybody can correct me, I accept positive criticisms,
Use Phusion polymerase from Finnzymes for cloning. It is the best cloning enzyme going around. It is a fusion between the DNA binding domain of taq with the proof reading domain of pfu, so it has high processivity and a very low error rate. The issue is that proof reading enzymes are good at reducing errors but they are slow and not robust where as Taq has good processivity and is robust but bad at reducing errors. Phusion gives you the best of both worlds.
I'm with you on that one, killerkoz- I absolutely love Phusion!!
NEB has another version called Phire-- I've just started using it and everything so far has worked!