How to identify the correct size of a large DNA band (>20kb) after being digested by a unique restriction enzyme?
I have recently digested a large plasmid with a size of 20,208 bp by XhoI, a unique restriction enzyme. I checked the fragment's size by running on an agarose gel 0.5%, 100 V, 1h. In theory, it should give a single band of 20,208 bp, and however, I received a single band with ~3kb. Why and how should I deal with that problem?
Thank you so much for any advice~
Best regards,
Hello Phan Thi Lam Hong ,
do you have an image of your gel ?
See this post for help !
https://www.researchgate.net/post/How-can-I-resolve-a-band-of-more-than-23kb-size-on-agarose-gel
If your band appear at 3kb, it could be wrong polymerisation of your 0.5% gel or your plasmid as not been digested and is supercoiled.
It seems very likely that your strain is contaminated with another plasmid, a 3kb plasmid is about the right size for a pUC or pBK type plasmid. The copy number of large plasmids is often much lower so it is possible that you just can't see it if it were there. But I see hints of a band around 25 kb range in the undigested samples. The digest lanes have that strange reflection making it impossible to see.
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