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Problem in the Digestion


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#1 zhgljj1998

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Posted 12 January 2005 - 10:57 AM

I used BamH1 and EcoR1 to cut pET-28a (5.5kbp), BamH1 is next to the EcoR1 in the plasmid. I tried to Double Digest and digest one by one. after ligation, no transformation colony. After checking everything, I thought it must be the problem of Digestion. is it possible that the plasmid like this (BamH1 Next to EcoR1) can be cut very well since we cannot see any differrence between double-cut and single-cut.

If we digest the vector by En one by one, Ecor1 first or BamH1 first, and need change buffer after the first En digestion? Please Help.

Edited by zhgljj1998, 14 January 2005 - 03:29 AM.


#2 vetticus3

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Posted 12 January 2005 - 02:16 PM

i double digest a similar vector with BamH1 and EcoR1, with no problems. I put both enzymes in together with y*/Tango buffer and leave it at 37'C for ~2-3 hours. Works fine.
how long is your digestion?
are you putting in enough enzyme?
can't think of anything else that could go wrong.
Try cutting with some other enzymes to compare.

#3 zhgljj1998

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Posted 13 January 2005 - 03:19 AM

i double digest a similar vector with BamH1 and EcoR1, with no problems.  I put both enzymes in together with y*/Tango buffer and leave it at 37'C for ~2-3 hours.  Works fine. 
how long is your digestion?
are you putting in enough enzyme?
can't think of anything else that could go wrong.
Try cutting with some other enzymes to compare.

Thanks. I tried to digest 2.5; 4 and 5 hr. Just the Enzyme and buffer i used were NEBuffer (Biolab). never used Y+Tango. `tried 1 and 2 microlitre Enzyme. no self ligation as well. Maybe ther is to much insert (3:1)?it made me mad for 2 months. I thank you anyway.

#4 zhgljj1998

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Posted 16 January 2005 - 10:26 AM

i double digest a similar vector with BamH1 and EcoR1, with no problems.  I put both enzymes in together with y*/Tango buffer and leave it at 37'C for ~2-3 hours.  Works fine. 
how long is your digestion?
are you putting in enough enzyme?
can't think of anything else that could go wrong.
Try cutting with some other enzymes to compare.

hi, man, thanks for your suggestion. I tried one more week, then, i quit to double digest with BamH1 and EcoR1 in the vector which is next to each other. just i am going to change other construct. i thought your double digest works well is amazing and lucky maybe. is there any special in your protocol?




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