Hello, I am currently working on plasmid cloning and today I got so confused about one of my colonies. After isolating their plasmids, NanoDrop results said that the concentration of DNA and A260/A280 ratio are so low so that I immediately thought somehow I contaminated the sample (or couldn't isolate the DNA properly) Then I prepared restriction diagnosis reactions and run my plasmids on agarose gel. I saw that the problematic plasmid runs faster than it should. The gel itself reminds me of supercoiled DNA but I really can't explain this result when I combine both data from gel and Nanodrop. Can anybody help me with this?
Thank you so much!
( The sample C3 has succeed so I added it as a reference)
I'm not sure but in can be differences in plasmid "conformation" between samples. Maybe samples which migrate faster are in more coiled and less relaxed conformation. You can emulate this situation using rope and rotate only one end but hold another.
Dear Elif,
I believe the concentration of C2 is more than C3 and so it looks like that. Also, you should rely on gel data than nanodrop. I usually dont believe on Nanodrop data, because if you take the OD three times of the same sample it never does tell the same value. They are varied always.
Thanks
Pratibha
A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms.
regards
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