Most important to know - effect of enzymes on DNA (Apr/17/2005 )

Dear All!
I just want to know that if I m using PCR product for clonning it will be always without Phosphate group at 5' end and a Adenine (nucleotide or nucleoside?) at 3' end if I m using Taq DNa polymerase.
What I m doubtfull is that the PCR prodct need to be treated by T4 DNA polymerase before the blunt end cloning into a vector which has also been treated with T4 DNA polymerase. I am not getting the exact chemical nature of T4 DNA polymerase treated ends.
Do they lack OH?
Do they have dntps?
Because what I m thinking that the PCR product and my vector has been treated by T4 DNA polymerase and I m trying to ligate them using T4 DNA ligase first from Roche and now from fermentas but ligation has even failed 10 times.
I needa help from all of you.. The thing i am planning wright now is to use T4 DNA kinase for my PCR T4 DNA polymerase treated product....
Looking for all of ur replies...
atif

your cloning scheme sounds complicated (too many enzymatic reactions). I have no experience with fill-in enzymes like Klenow and T4 polymerases because I design my schemes specifically to avoid 'complicated' molecular biology.

t4 pol functions as a 5' -> 3' DNA polymerase and a 3' -> 5' exonuclease, but does not have 5' -> 3' exonuclease activity.

blunt-end ligations are tough enough, but add onto that having no 5' P is making it twice as hard. T4 Ligase only halfway completes the job of ligating a raw pcr product onto your vector (because of the missing P). The nick is repaired once it goes into the bacteria.

If you can obtain vectors like pGEM, etc., which can directly accept PCR products with 3' A overhangs, you can directly ligate your PCR pdt into that. The "sticky end" created by the single A overhang is much better than having a completely blunt end.

You can also design primers that will have a useful restriction site which you can directly utilize to subclone into a vector.

How long are you doing your blunt end ligations?

your cloning scheme sounds complicated (too many enzymatic reactions).  I have no experience with fill-in enzymes like Klenow and T4 polymerases because I design my schemes specifically to avoid 'complicated' molecular biology.

t4 pol functions as a 5' -> 3' DNA polymerase and a 3' -> 5' exonuclease, but does not have 5' -> 3' exonuclease activity.

blunt-end ligations are tough enough, but add onto that having no 5' P is making it twice as hard. T4 Ligase only halfway completes the job of ligating a raw pcr product onto your vector (because of the missing P). The nick is repaired once it goes into the bacteria.

If you can obtain vectors like pGEM, etc., which can directly accept PCR products with 3' A overhangs, you can directly ligate your PCR pdt into that. The "sticky end" created by the single A overhang is much better than having a completely blunt end.

You can also design primers that will have a useful restriction site which you can directly utilize to subclone into a vector.

How long are you doing your blunt end ligations?