2 replies to this topic

[gulsatar]

Hi friends

I have pBluescript vector and e coli which I made by myself. I used Onetaq (NEB) for my pcr reaction. I used degenerate primers and I dont now full sequens of my sample, neither enzym cut site.  
My questions

1.   should I do any treatment to my pcr product before ligation
2. I dont know enzym cut site of my sample. If I use EcoRV for blunting end. Does it work.
3. If I directly ligate my pcr product to vector. what happens. Does it work


Thanks

[Papaver]

Hi,
do you know if the PCR-product is already blunt end? The common Taq polymerase produces an A-overhang, other Polymerases not (like Phusion). I once used this for blunt end cloning into a blunt cutted vector.

[Kamran]

Q8: What type of DNA ends result from a primer extension reaction or a PCR using OneTaq® DNA Polymerase?

A8: A sufficient proportion of PCR products generated using OneTaq® DNA Polymerase contain dA overhangs at the 3´end to enable ligation to dT/dU-overhang vectors.
http://www.neb.com/n...tM0480.asp#2167



The best option is to TA clone your PCR product however, you may also do blunt end cloning after phosphorylating your inserts termini (if you are not using phosphorylated primers)