I have cloned two domains of a gene into two bacterial vectors using cut and paste cloning with different RE sites. I checked the putative clones by colony PCR and Restriction Digestion (fallouts expected). Both seemed positive as the DNA bands were observed at the correct expected sizes on agarose gels. I then inducted the genes by IPTG and observed protein expression at correct sizes on SDS-PAGE gels. However, the Sanger Sequencing results of the plasmid samples seem to be negative for the clones. The results have good peaks(reads) but the sequences do not align with the gene of interest.
I am confused as to what could be wrong. I am looking forward to suggestions or any other experiment to check the matter.
Thanks in advance.
Unless there was some mix-up in your clones, the most likely explanation is that your clones contain spurious DNA inserts that happen to be the right size, but are unrelated to the desired target gene. You could get more information by starting a BLAST comparison with the Sanger reads to see if they match any sequence in GenBank. Otherwise, I am afraid that you will have to redo the cloning. In doing colony PCR screening, try to use at least one primer that hybridizes to the insert and not the vector. Likewise, when checking the restriction pattern, include a restriction enzyme that cuts within the insert. Finally, before using a clone for protein production, make sure that Sanger sequencing of the clones match the expected sequences.
Unless there was some mix-up in your clones, the most likely explanation is that your clones contain spurious DNA inserts that happen to be the right size, but are unrelated to the desired target gene. You could get more information by starting a BLAST comparison with the Sanger reads to see if they match any sequence in GenBank. Otherwise, I am afraid that you will have to redo the cloning. In doing colony PCR screening, try to use at least one primer that hybridizes to the insert and not the vector. Likewise, when checking the restriction pattern, include a restriction enzyme that cuts within the insert. Finally, before using a clone for protein production, make sure that Sanger sequencing of the clones match the expected sequences.
Agree with @Pierre. Since you have checked at every point of your express., I suggest you to first try aligning the reverse complement of the Sanger sequence with your reference goi. You can do a quick blastx (protein db search using translated nucleotide seq.) search too to make sure that the sequence is not what you ar expecting.
Best wishes
Another possibility is that you have mixed plasmids (or some plasmids with your insert and some without). So as a mixture you see the band you want and the protein you want but not by sequence. Or if you have retransformed you just picked the wrong colony. Mixed plasmid preps after cloning are quite common.
Or maybe it is as simple as someone grabbed the wrong primer for your sequence runs. Sometimes it is just something simple.
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