We studied somatic mosaicism in neurons. We designed specific primers for a neuronal gene that should produce a long product (1700 bp) and performed a PCR with genomic DNA and LongAmp polymerase. Each neuronal culture sample had a distinct set of byproducts. We tried to isolate and sequence them. When we cut a product about 800 bp from the gel and performed a second round of PCR with the same primers, using 800 bp fragment isolated from the gel as a matrix, somehow the product length was 1400 bp. We sequenced this product and it turned out the primer annealing was specific and this product comes from the gene we studied, but this was a variant with a 300-bp deletion. However, I can't wrap my head around how the 800 bp matrix could produce this fragment that is longer and is not a result of polymerization of any 800 bp sequence. Is it possible that somehow a small portion of a longer product migrated on the gel together with a shorter product?
It is unlikely that a unique 1400 bp fragment co-migrates with a sequence that is 800bp long.
It is possible that the 1400bp sequence could have been amplified from a contaminant in your work area. You can do a negative control reaction where you omit the template and add ultrapure water instead. To be safe, you can clean your pipettes and wipe down the bench and centrifuge. Try to use fresh buffer, water and tips. Change the polymerase if possible.
- Badges
- Science method