Blunt end cloning of 4Kb product - (Aug/18/2011 )

Hello everyone,

I need to clone of my gene (around 4kb) into pIRESneo3 vector. So I designed & ordered primers (Fwd primer with NheI site & Rev primer with BamHI site). The problem is I forgot to add additional bases (GC clamps) before these enzyme sites. So I wanted to check whether these enzymes cut the PCR product or not. I digested 0.5ug of gel eluted PCR product at 37⁰c for 4 hrs (Fermentas enzymes)1) Double digestion2) BamHI alone3) NheI alone

Then I self ligated these products with T4 DNA ligase from Fermentas at 22⁰c for 30 min.
I ran the product in a gel. But I didn’t see any concatemers (only single band was there).

MCS of my vector contains blunt end cutter EcoRV. Should I ligate undigested product or design new primer?

Please share your experience & suggest me something…

Thanks in advance,
Sen

I doubt very much that these cut. You need to purify the digestion product with BamHI, because the enzyme cannot be heat killed, so the enzyme will remain and re-cut the ligation product (but I doubt if this was your problem). I would definitely re-order primers, but you could try a TA cloning reaction or blunt ligation. I don't predict an easy success.

The enzymes are almost certainly not cutting and I would also get new primers.

For blunt end cloning you would need to phosphorylate your insert and dephosphorylate the vector but would your insert be in frame?

phage434 on Thu Aug 18 22:54:16 2011 said:

prodes on Fri Aug 19 20:23:55 2011 said: