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Slight Smear after Digestion - (Jul/28/2010 )

Hi,

I try to clone my TOPO Insert in another plasmid.

When I digest either the TOPO or the other plasmid I get smear. I tried NEB and completely new Fermentas Enzymes and used filtered pipets under the clean bench when pipetting. On the picture you see the plasmid I wanted to cut. My Gelpicture:

(M | KpnI | PstI | XhoI | HindIII | HindIII + Pst I | Pst I + KpnI | Digestion without enzyme but buffer | Marker)


Sure you see a cutted band but there is a smear above and under the band. Ligation didn't work with it.
A friend of me did this digestion for me and there was smear on my digestion but no smear on his digestion. So I made a new plasmid isolation (Fermentas) and there was also smear.

Thanks in advance

Mara

PS:
Restriction:

16-17 Ál Plasmid Isolation (50 ng * 16 Ál)
2 Ál FD Buffer
1 Ál per Enzyme

or larger scale

43 Ál Plasmid Isolation
5 Ál FD Buffer
2 Ál per enzyme
Attached Image

-MaraJade-

Likely the problem is contamination in your plasmid prep. Almost all of the volume of your digest is from your plasmid, so there is essentially no dilution of inhibitors in your reaction. Try the reaction again, cutting 3 ul of your DNA (150 ng) in a total of 20 ul. You'll dilute any inhibitors by 7x this way, and the reaction will most likely work. Common inhibitors include alcohol from the miniprep and Gu-HCl from the miniprep. Make sure you are spinning columns completely dry prior to elution (and also dilute inhibitors when doing the RE digestion by making the DNA a small component of the reaction).

-phage434-

Thank you very much!! I did digestion with the new approach and it looked very nice. After Ligation and Transformation I have some clones with Insert in the colonyPCR

-MaraJade-