Hi, I am wondering why DNA smear in this gel. The plasmids extracted from stbl3 are cut with restriction enzymes. Is it possible for overloading, nuclease contamination, or genomic DNA contamination? (May the contamination of bacterial debris as performing mini-prep contribute to it?) If so, is it possible to identify it or avoid these unwanted results? Thank you
hello,
Basically there are multiple factors which disturbs the Nucleic acid integrity like,
1. Genomic contamination with phenols, proteins and other debris for these go through proper instructed extraction protocols.
2. Gel contamination or gel comb contamination, for these clean up those combs and trays so that it shouldn't bear saline or salt load.
I suggest you to clean those combs before you use, because overloading or well feeding won't disturbs the gel profile.
I think the problem you are having likely stems from the fact that stbl3 is EndA+. Most cloning strains have the EndA- allele in them which inactivates the periplasmic nuclease from E. coli. So stbl2 is EndA- but not stbl3. You could 1) be sure your mini prep protocol has the appropriate steps to remove EndA; 2) changes strains; 3) do NOT treat your mini preps with RNAse (the RNA acts as an inhibitor to EndA); or 4) don't worry about a little smear on your gels. I would just go with 4 and not worry about it.
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