I am cloning a gene into a pET28a vector. I used NEB enzymes NheI and HindIII to to double digest my insert and the vector using 2.1 NEB buffer. Incubation was done at 37 degree overnight (10 hours). Gel extraction was done for both double digested insert and vector. The digestion looks clean and clear in the gel. This was followed by ligation at 16 degree(10-12 hours).
During transformation a control was set up with double digested vector in XL1 blue cells. I found a number of colonies after incubation, Whereas blank XL1 blue cells did not grow on kanamycin plates as per expectations.
This shows un even double digestion of the vector.
How can we improve this scenario to overcome the ambiguity over cloning results?
Hi Gaurav,
Use CIP treatment, dephosphorylation of cloning vector DNA to prevent recircularization during ligation.
How close are these restrictions sites sitting next to each other ? I Think the problem is that after the cutting of one site the other one doesn't have enough room for the restriction enzyme to attach at the site. There are guidelines from NEB as to how much extra nucleotides should be present next to the restriction site when it is at the end of the linearized plasmid. I think this the case for you also.
Try to set up two extra controls where you are digesting your plasmid with only one RE and see at what efficiency they cut. I think its a matter of RE cutting efficiency. And when we have these double digestion the efficiency of one enzyme is masked by the efficiency of the other one and you anyway see linear plasmid.
Thanks alot for your feedback. I guess, It's a case of incomplete digestion by one of the enzymes.
DO sequential digestion.
Choose the first restriction enzyme digestion judicially by seeing the buffer composition so that you can avoid precipitation (if possible).
OR
1. Do digestion sequentially
2. set two independent set
i) Restriction enzyme NheI
ii) HindIII in other
3. check (aliquot, may be 1/10th of total volume) the digestion by Gel Electrophoresis (while continuing the digestion)_
4. then add other enzyme like HindIIi in tube (i) and NheI in Other (ii). make sure about the buffer compatibility.
So you can have an idea which one is wrong. If both are OK, then mix them.
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