I have cloned a Bacillus gene in E.Coli pET20b vector at EcoRI and HindIII sites. The vector was already having a gene cloned at the same restriction sites, therefore, before ligating the bacillus gene, the vector was digested with EcoRI(HF) and HindIII restriction enzymes so that the previously cloned gene can be kicked out of the vector. The digested (EcoRi and HindIII) PCR product was then ligated into the digested vector and positive clones were obtained which was confirmed by digestion with EcoRI (HF)and HindIII. The double digestion showed the fall off of the insert from the vector, however, the sequencing results does not correspond to the desired gene sequence. The sequence shows the gene of some E.coli strain which has similarity with the previously cloned gene as per the BLAST results. Moreover, the sequence does not have the primer sequences present and out of five recombinant plasmids sequenced, only one plasmid has the His tag sequence and others don't have. I have performed this twice and end up with the same wrong result. Is it problem of contamination or wrong primer sequence?
Dear NAmrata
just a question: did you performed the gel extraction of the digested and defosforilated vector before to use it for the ligation with the new inserts?
Because i think that this have to be done, to minimize the problems due to re-ligation of the digested insert or strange insert rearrangment.
Of course To provide an opinion regarding primer sequence, we need to see the primer and the gene sequence.
As generall comment, i would like to suggest you to move to an enzime free approach, as the PIPE cloning, which is appliable to any vector, do not require any enzime digestion and if coupled with PCR colony screening can provide you very fast and reilable results.
You can find more details about it on my blog: http://proteocool.blogspot.com/ at PAGE1 Gene cloning
good luck
Manuele
Namrata Chakravarty
- Was that your purpose to clone one gene in place of other? Why did you do that?
- If you have confirmed that you have to right DNA fragment, sequencing and homology should not be much different. There must be something wrong with the fragment preparation in first place and it got magnified in the later part of the experimental process.
Dear Manuele Martinelli
Yes, I performed the gel extraction of the digested vector before to use it for the ligation with the new inserts. I will consider your suggetsion for enzyme free cloning. Thank you.
Abhijeet Singh yes, that was a purpose to clone my gene of interest in place of the previous insert. Both the genes encodes for same enzyme but one is from E.coli and another from bacillus, therefore there is differnce in their sequence.
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