I am trying to clone my targeted gene by replacing GUS site of pCAMBIA1301 to utilize the 35S promoter. For that I have used 2 restriction enzyme i.e. NcoI and BstEII, and both the enzymes are site specific.
But when I tried to double digested the pCAMBIA1301 vector to retrieve the backbone of plasmid without GUS, the NcoI enzyme is digesting an unspecific site yielding one more band of about 1.9kb other that the probable Gus (2 kb) and backbone vector (9.9kb).
I have tried 1 , 2, 3 hr incubation but nothing seems to work. In all condition, the result is same.
Can anyone suggest me a solution
I am using NEBHF NcoI and BstEII.
buffer : 10x r cut smart buffer.
Can you show your gel results and tell us what ladder you're using? Usually it's tricky to distinguish between bands >1500 bp that are so close in size.
Is the digest in your picture only NcoI alone or double digested with NcoI + BstEII?
Regardless, clearly there is a problem with the vector. My suggestion would be to get the vector again from a fresh source.
It is a double digestion with both NcoI and BstEII.
Thank you for your suggestion.
Paromita Saikia
Are you successful in doing restriction based cloning in pCAMBIA 1301 by replacing GUS site ?
So can you mention what was the problem and how you have resolved the problem?
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