RF-Cloning.org Forum

Hi Steve,

hope you are doing fine.

I am trying to insert a 1.5 kb gene into a 3.6kb (pPICZalpha A) vector. I have designed primers accordingly, each with about 25 nucleotides from gene and appropriate site in vector. My first PCR (for amplifying gene) works very well and I get single band. After doing PCR purification of the product I use it for RF cloning PCR using pPICZ alpha A empty vector as the template. As I could not get my PCR working using 200 pM vector and 6 nM  insert (from van den Ent et al., 2006 paper), I read your blog and did a 2 step PCR's using 400 and 600 pM vector and 10 nM and 12 nM insert respectively. However both these PCR's did not work.

Also, in a separate experiment, I'm doing another cloning where I'm inserting a gfp gene into a pHIS17 vector (2.6 kb) that already has my gene of interest (1.5 kb) (Please note that I'm inserting this gfp gene at a specific site in the 4.1 kb clone containing my gene of interest). Here also, the RF cloning PCR does not work.

I use Accuprime pfx polymerase for these PCR's and hence the experiments are designed considering extension rate of 1kb/min.

Can you please help me sort out my cloning issue? 

Thanks
Shrikant