Hi all,
I am working on freedom p CHO vector. There are two restriction sites called
AVR II/ Bst Z171 and ECO RV/Pac I. I am able to digest my vector at the sites and I am able to clone at any one site with my insert. But the problem starts now as I am unable to ligate the other insert even though primers for insert and digestion is proper. I have used infusion cloning and also normal ligation methods.
Can any one suggest me some tips.
Thank You...
Just did some checking and your AVR II and PAC1 sites are not compatible. The BST and ECO RV sites are both blunt but the over hangs of the other two are not..
I don't think that AvrII C|CTAGG and PacI TTAATT|AA are compatible for cloning unless i am missing something
Hi,
I agree with Daniel you seem to have four different sites here, and none of them are compatible cohesive ends or isoschizomers (NEB has some nice charts for this https://www.neb.com/tools-and-resources/selection-charts/compatible-cohesive-ends-and-generation-of-new-restriction-sites and https://www.neb.com/tools-and-resources/selection-charts/isoschizomers, respectively).
I took a peek at the freedom p CHO 1.0 vector and it seems as there are two promoter sites you can clone after either the CMV/EF1 promoter (which has AvrII and BstZ171 after it) and a EF2/CMV promoter (which has EcoRV and and PacI) after it.
So depending on which promoter you were aiming to insert your gene after you would go into either AvrII (5' end of insert) and BstZ171 (3'end of insert) or EcoRV (5' end of insert) and PacI (3'end of insert). Is this what you are doing? I'm a little unclear from your question since you said you could clone at any one site, but your second insert was giving you problems.
Does anyone has sequence of pCHO 1.0 vector? I'd like to know it before committing to buy the whole kit.
Thank you
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