After restriction digestion my bands just disappear - (Dec/02/2004 )
I need some urgent help here guys....
I did mini-prep of my plasmid clones using alakali-lysis method...yield close to 400- 700 ng/ul... good bands on the gel
Now to identify poitive clones I did restriction digestion with pvuII for one set of clones and HindIII for the next set. As control, I also digested a vector.
and I ran the digested plasmid side-by-side to the undigested ones....
and the digested ones have just disappeared: all i see is a smear-type band at the bottom of the gel. The undigested ones are fine and the control was also cut.
Is it that I have some nucleases in my miniprep or what?
Has anyone else faced this problem...I'd appreciate some help here
Have you managed to solve this problem yet? Im having a similar problem but I am doing a single cut with HindIII. I ran the cut plasmid and had a strong linearizedd band and a smear, then ran the sample again the next day and its all gone. Not sure what to do about it, this problem has appeared multiple times (always with HindIII...different tubes of it too) using different preps of the same plasmid. Im using the right buffer and I heat inactivate after 1hr. Its getting very frustrating!
Poxvirus I would say that you have a nuclease contamination problem in your plasmid or buffers. If it is only happening with HindIII and on different lots then the likely source is the HindIII buffer.
"...and the control was also cut."
So the vector-only digest was not degraded, but was cut correctly by the enzyme(s)?