Size of my gene of interest cloned into pet 28 alpha 6857bp. After transformation I did plasmid digestion with ingle enzyme xhoI. But the single band size is not equal to total size 6857 instead the band size is near to 4500bpb. I don't know why this happen kindly picked 18 transformants and did a colony PCR for my insert, 2 came out positive. I did miniprep on those two and a diagnostic restriction digest with the same enzymes, buffer, conditions etc. as used for my cloning, but when I ran the gel I just got one band at the top (undigested plasmid) and another at ~3 kb. Nothing of the correct size for my insert or the plasmid... has anyone else experienced this? What should I do ? Please pray
There is a single site for XhoI in pet28a, but what about your insert ? A possibility is that the enzyme recognition site for XhoI is present multiple times within the insert, leading to the formation of multiple smaller fragments. Additionally, it could be that the colony PCR was done on a plasmid that does not contain the insert. It is it also possible that the 4500 bp band is a supercoiled form of the plasmid. Supercoiled form of a plasmid migrate faster than linear form, and linear form faster than nicked circle form.
Sir ! There is a single site for XhoI in my gene of insert. But the band size near 4500bp for single digestion.I didn't perform any PCR. I did my work from the direct culture, and did plasmid extraction and run on gel after confirmation did restriction analysis. But the band size for both XhoI and MluI were different. MluI cut on double site with in my gene of interest and within the plasmid, two cutting sites with MluI.
Colony PCR is known for it's very high rate of false-positives, especially if both primers anneal to the insert sequence. You're very likely to amplify any unincorporated/unligated insert.
Use one primer that is in the plasmid backbone and one that is in your gene of interest.
Good luck!
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