I have a problem with my cloning, I'm trying to clone a 483 pb in a pSecTag vector with restriction sites HindIII and BamHI. Here are the procedures I'm doing:
• I introduce the restriction sites BamHI and HindIII into my insert by PCR with Pfu (blunt end polymerase) and gel purified it.
• I clone my insert in a pJET1.2/blunt vector. I get enough colonies and I tested few of them with an internal insert primer and a vector prime via PCR. The results are positive.
The problem came when I digest my insert from pJET and try to clone it in a pSecTag vector.
• I digest my insert and the vector with HindIII and BamHI in Buffer Red during 1.5h at 37ºC (fermentas). I purify the insert and the vector from the gel and checked that all fragments run at the correct size on a gel. All correct.
• I do the ligation (T4 DNA ligase) using a 5:1 Insert:vector and less tham 100ng of total DNA. The ligation (20ul) is performance at 22ºC for 30 minutes. I get few or not transformant colonies. When I get colonies, I tested them by PCR:
** • With internal primers, the result are positive
- With a primer for the vector the result is negative.
- The strangest thing is that when I use pJET primers that do not anneal in the pSecTag I get positive results.**
For transformation i'm using chemocompetent DH5-alpha, with a heat-shock of 42°C at 45 seconds. I tested the bacteria with a plasmid before using them.
I change condition several times but always obtain the same result. I follow all the protocols but I don’t get my construction.
Anybody knows where the problem is? Thank you.