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Probelm with double digestion

Probelm with double dige

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#1 shaajahan7



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Posted 15 February 2013 - 04:09 AM

Dear Friends,
I have plasmid DNA and i wanted to separate my vector and my insert. I tried EcoR1 and BamHI (From NEB) with NEB 2. And i tried 30 ul reaction and here below i am giving reaction volumes:

10x buffer 2ul
BSA: 0.2ul
Water: 7.3ul
BamHI:1 ul
pDNA: 8ul

O/N incubation. For quality of digestion i used 0.8% Agarose gel and i loaded my samples along with ladder. I got clear ladder and i got plain smear my samples.

Can anyone help me get out from this problme

#2 cellthetruth



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Posted 15 February 2013 - 07:02 AM

pay attention to these points, try again and see what happens. most probably the result will be good (or easier troubleshooting possible)
1. at which temp did you digest o/n? 37°C is to much, you'll degrade your plasmid (BamHI has star-activity under certain conditions). 2 hours is enough.
2. how much plasmid did you use? in ng/uL, not only the volume.
3. why do you use buffer 2? BamHI comes with buffer 3, EcoRl has a unique. Use buffer 3.
4. did you precipitate your plasmid prior to digestion? smear may be a contamination.
5. how do the controls look like? single digest with each enzyme as well as the uncut?

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