I did a lot of cloning using the gibson assembly master mix and every time it worked perfectly. I must have done more that 30 cloning with great results.
However, now I've been trying for the last month to do a routine cloning and I only get clones where the backbone closed on itself.
I want to clone two inserts (1 of 450pb and one of 900pb) into pcDNA3.1 digested with XbaI and BamHI. I purified my inserts and my backbone on agarose gel. I do the cloning with 75ng of vector and and 3-fold excess of each insert.
I tried to do the cloning reaction with only the backbone ( no insaerts) and I also obtained a lot of colonies. How is this possible? What should I do. I already tried to make a new prep of digested pcDNA3.1 with no sucess.
Thank you
Hi there,
Obviously there is a problem with the vector double digest :
Have you checked the efficient linearization of the vector with each enzyme individually?
Do you gel purify the double digested vector?
If yes to both questions, you shouldn't get any clone with the backbone alone...
The BamHI may exhibit star activity if the digestion time is too long.
Sounds like you're doing it right. It happens on occasion that certain assembly sites do not work. Change them...
If at all possible I prefer to make the vector by PCR. It lets you skip some of the more fragile process steps and makes it easy to change things on the fly, without being constrained by where the restriction sites are. Order a couple of different primer pairs that do what you want right out of the gate, and see what works. You need to use minimal template (1:100 or 1:1000 of a regular miniprep) to prevent vector carryover, and a fusion polymerase.
Hi! Was wondering if you solved your problem, Xavier? I am having a similar situation now - Any help/suggestion would be good. thanks!
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