I did cloning of vector containing a promoter sequence, confirmed it by PCR and plasmid size. Then transfered the gene to expression vector and sent it for sequencing. Whet I got sequencing results, the sequence was not of promoter of interest it is of some mRNA and not of the vector. I tried to locate primers in sequencing results but did not find both. Then I came to know that my vector did not contain the sequence of those primers used for sequencing. I am surprised then what is sequenced. Can anyone tell me what was amplified if it is not the vector or promoter sequence?
You state that you sent your insert off for sequencing. It would be best to contact whomever you sent the insert to, and discuss it with them.
This is a very confusing post. Can you please share what vector(s) you are using and what time of sequencing oligos (designed against vector or insert) you used?
Thanks all for your response.
Raquel Rabionet Yes I had contamination during PCR before cloning. My desired product size was 1180 bp but there were three more bands. I cut and purified the desired fragment, then cloned. No sequencing was not done at that step. Vector was purified for sequencing.
I am using Gateway expression vector pHGWFS7. Universal primers M13 -20 F and R were used for sequencing. Later came to know that vector does not have binding site for these primers. Confusion is that if vector does not have binding site then what is sequenced then? Peaks are very clear.
M13 sequencing primers are not always listed on plasmid maps. Do you have the sequence for pHGWFS7 and have you checked it directly for M13 sites? If they are truly absent, then most likely you cloned a different plasmid by mistake. For Gateway cloning you need to make sure that you are using an F' negative E. coli strain that is ccdB sensitive. You also need to make sure that you have a way of removing the plasmid that encoded your promoter sequence (assuming that you one this instead of genomic DNA for template) prior to the Gateway recombination step. Otherwise, you will simply recover this original plasmid in your cloning.
How have you verified your plasmid is pHGWFS7?
hi, I am facing somewhat similar problem. I had PCR amplification for a fusion gene by using cDNA. when I got the sequencing results, it doesn't matches to either of the 2 genes (I used primer pair belong to 2 different genes as Forward and reverse primer). When I blast the sequencing result, it belongs to a 3rd gene. I again checked the primer specificity and find it specific for the 2 genes. What would be the possible reason for getting sequencing result related to a 3rd gene???
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