I've been trying to clone a gene from H. influenza. Every step (PCR, digestion, ligation) seemed to work based on agarose gel images. Before I sent the DNA for sequencing, I digested the plasmids recovered from transformed cells and run the digest on agarose gel. There were 2 clean bands that appeared at the right size, 1 for the vector (~3.3 kb) and 1 for the insert (~ 1kb). There were no additional bands on the lane. But the sequencing results came back that I got a totally different species (E. coli). The discrepancy is totally confusing. Any ideas of how/ why this could have happened besides contamination?
Hi,
Have you analyzed the E.coli sequence in your plasmid? Is the sequence similar to your H. influenza gene? Are there at least similarities at the ends?
You could check the initial colonies via cPCR using gene specific primers. This method should be used in addition to the restriction digest. It provides information about the sequence of your insert and not just the size of your insert. A combination of both methods could improve your clone selection prior to sequencing.
Best,
Boas
Wich species was you using as receptor for the cloned plasmid?
Boas,
I did not run cPCR as I did not work with a large number of clones. I thought the advantage of cPCR is that it allows you to screen for the presence of plasmids in competent cells, essentially the same idea as doing a miniprep. But I will probably give it a try. Thanks for the suggestion. :)
Hi again,
Many colonies could by screened in a cPCR with less afford and time than by plasmid isolation and restriction digest. That is the main advantage. Moreover, this method is specific if you use appropriate primers. Unfortunately, you cannot exclude the presence of other (additional) plasmids. Therefore, you should keep doing a plasmid isolation and following restriction digest with promising clones after cPCR.
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