I have an insert size of 900 bp, trying to clone it in pET28a vector. But after every the transformation when i am doing colony pcr and digestion check i can only get the fragment of 500 bp. Why is it so? The restriction sites are BamHI and SacI and the gene doesn't have internal sites of these, which i could know by sequencing. So why is it so. Do i need to subclone instead of directly trying to clone in pET28a plasmid?
Sucloning is generally not required. I'm not sure what is exactly happening since cloning is a multistep procedure and problems can happen at any step. You can check the ligated product by PCR using gene-specific cum vector-specific primers. You can do the same with colony PCR. Possibly there might be an issue with ligation. The ligation step is critical in gene cloning.
Good wishes!
Is the 500bp band just vector or is it an incorrect insert?
If it is an incorrect insert, then most likely the problem is either that your reaction is contaminated with pET28 carrying a 500 bp insert, or that the 500 bp is derived from your 900 bp for reasons that are not clear. You could sequence one and see what it is.
The choice to subclone often has to do with how precious or heterogenous your template samples are. Have you run your restriction digests for cloning on a gel? Are your restriction enzymes still before their expiry date? Sometimes performing an overnight digestion can help. Are you using a CIP treatment step or not?
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