RF-Cloning.org Forum

shrikant

Member

Registered: 2016-04-30

Posts: 1

Problem with RF cloning

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

Last edited by shrikant (2016-04-30 19:55:19)

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Problem with RF cloning

Hi Shrikant,
I assume you designed your experiment manually? I'll get you to do me a favor and use the RF-Cloning site to recreate the projects, that way I can look at them more closely. Either save the projects to your account (I can pull them up from the back-end) or send me an email with their URLs.
Hopefully we can figure this out
-Steve