I am planning to do some gateway cloning and I have all the pENTR/TOPO vectors with attB sites. What I need to know is how to add these attB sites into my vector so I can easily exchange my gene of interest from the gateway clone into my plasmid of interest. I hope this sounds ok.
Hi Naushad,
I did some gateway cloning. To insert my "region of interest" into my vector, I amplifyed the region with primers on which one a add the attB site... I purified my PCR product and after I can to the gateway cloning.
If you need help, I can give you more information.
Thank you Celine. More information would be of great help. I was thinking of adding polylinker with MCS into my vector and then digest the vectors (one with polylinker and one with attB sites) and ligate them to generate attB sites into my vector. I m not sure if this would work as i just get to know that the digestion is producing blunt and sticky ends together.
HI Celine, Thank you for the answer but I should clarify on more thing. My vector does not have any attB sites. First I would need those sites and then I would clone my gene of interest into the vector (with attB sites). i want to generate those sites first
First of all pENTR/D-TOPO vectors are entry vectors so they have attL sites and you can clone it in without having attB sites in gene specific primers. Rather you need a 4 bp sequence at 5' end of the forward primer to maintain direction in TOPO cloning. If you want to go with pDONR series vectors then you need attB sites in primers as pDONR entry vectors have attP sites.
Hi Naushad,
Have you find the answer?
1. All you need is Gateway Vector Conversion System with One Shot ccdB Survival 2 T1R Competent Cells kit.
2. If you already have a pDEST vector of some kind, you can excise or PCR the attR1-Cm(R)-ccdB-attR2 cassette from it and insert it into MCS of your vector of interest. You need a ccdB resistant E. coli in this case also.
- Badges
- Science topic