Hello!
We have designed primers, which were used for a PCR reaction. We obtained a PCR product size of about 1180 pb (even for the positive control). when sequencing those PCR products, they correspond to the cibled gene.
The problem is when I check the insilico expected size either by alignement or other programs among which (http://www.bioinformatics.org/sms2/pcr_products.html). The expected size was completely different (it was about 1395 pb?!
Can someone help me to solve that?
what species are you working in?
I didn't get a pcr product against the human genome, so i tried to allign your imput sequence to mouse and human genomes, and got no real hits...
did you sequence your pcr product? does it make sense at all?
I am working on bacterial species (Bacillus thuringiensis). after sequencing the products, they correspond to the cibled gene. The only difference is the size of PCR products. theoriticly, it should be 1395 pb, but after performing a PCR reaction and running an agarose gel, the obtained size was about 1180 pb!
By all means check and double check everything but never forget that your PCR product may be shorter because it is not the same as previously published sequences. Don't always fear that things are wrong they can be different and this is the beginning of a very interesting study.
In accord with Artur Byrzynski, it would really help to know more about the experimental details. What are the sequences of the primers? Did you sequence the complete PCR product? What primers did you use for the sequencing? What is the experimental sequence? Did you align the experimental sequence with the "in silico" product sequence? You can upload files that include this information.
One possibility is that your agarose sizing is not correct. Another possibility is that one of your primers is very similar to a sequence that lies within the intended amplification region. In that case, you would generate a smaller product that is missing a terminal segment of the desired amplicon. Further distinction between these possibilities or consideration of others would require a detailed review of the information requested above!
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis
- genetic techniques
- Sequencing