I am looking for pTRIPZ vector with a resistance other than puro.
I would appreciate if someone that built it already could share.
I tried to replace PuroR by HygroR using Gibson assembly but my PCR for the vector (with PuroR deleted) keeps failing using Fusion or Accuprime (12kb).
I attach the map and would appreciate any advice on an alternative cloning strategy.
Thanks,
Igor
I haven't worked with TRIPZ directly, but in analogous cases I have been successful using QuikChange from Agilent.
I would first remove the pac gene using QC "deletion" protocol, and then insert the whole hygro (or similar) resistance gene using QC "insertion" protocol. I have used the QC kit to introduce fragments up to 1.2kb, with good results.
Good luck.
http://www.chem.agilent.com/Library/applications/5990-5152EN.pdf
http://www.genomics.agilent.com/files/LitItems/QuikChange%20II%20Large%20Insertions.pdf
http://www.ncbi.nlm.nih.gov/pubmed/?term=An+efficient+one-step+site-directed+deletion%2C+insertion%2C+single+and+multiple-site+plasmid+mutagenesis+protocol
Thank you, Andrea.
I thought that Quick change also relies on PCR of the whole vector plasmid?
I have troubles to get this to work (probably because it is 13 kb).
Do you think that the enzyme in QC is better than Phusion or Accuprime?
Yes it relies on PCR, too. But it is a simpler protocol, in that it does not require excision or ligation (the ligation is performed later). I have used the XL version that is optimized for longer plasmids (our are also 12-13kb), the enzyme they use in the kit is supposedly optimized for processivity of long DNA molecules. It works for me (us).
Just a thought, you did include an exonuclease in your reaction, right? Because only the Accuprime has a 5'-3' exonuclease activity that is required for Gibson assembly.
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