Check the primers you are using for sequencing are in the correct direction, especially if you are not using standard M13 forward or reverse etc. It is quite possible your sequence trace represents vector sequence. Have you checked if there is a fragment insert first by restriction digest?

Alternatively, trim the first & last ~100 bp from your sequence and try BLAST'ing that.

Check the primers you are using for sequencing are in the correct direction, especially if you are not using standard M13 forward or reverse etc. It is quite possible your sequence trace represents vector sequence. Have you checked if there is a fragment insert first by restriction digest?

Alternatively, trim the first & last ~100 bp from your sequence and try BLAST'ing that.

I agree with Kevin. If you confirmed the insertion of your gene of interest in the plasmid before sequencing, you should get it.

agree.

Doing a pcr check of the plasmid before sequencing would also help

Yes, check the expected size of the insert with PCR first. If the cloning is directional, use one primer for the cloned sequence and another for the vector. If the direction may be both ways, use two vector primers instead. You can also check the clones before making plasmid preps. Just use some of the bacterial clone directly as a template for PCR. 

Hi Arun,

I hope the above answers solved your problem. If not you may better tell us in more detail how the cloning was done. In case you isolated your insert from an E. Coli DNA prep one of your positives may by chance contain genomic E. coli DNA. In this case, do a restriction analysis of your positives including an enzyme that cuts your insert. This should identify your human derived insert.

We have encountered similar problems. 1st example, we tried to clone 5'-end of yeast YAP1 mRNA by RACE. It turned out that cloned fragment belongs to some enzyme from Marinobacter spp. The problem was in the forward primer, its RNA moiety was degraded (may be it was incorrectly synthesised). I would suggest to check the integrity of primers by PAAG.

2nd example, we tried to clone one of subunits of yeast CCT shaperone complex. The cloned fragment corresponded to one of E.coli proteins. We noticed that target PCR fragment was in very low concentration. Perhaps the DNA used as matrix for PCR was contaminated with E.coli genome and PCR conditions or primers for the target fragment were not optimal. I would recommend to check the presence of secondary structures at the sites of annealing primers.

I think you need to provide more information--what is the Ecoli match? Did you sequence more than one clone? Did you clone in Ecoli (likely) or another bacterium?

I suspect the E. coli DNA comes from one of your reagents. Most enzymes are prepared from recombinant E. coli and they almost always contain traces of E. coli DNA.