I am working on head and neck cancer samples and trying to amplify a specific gene of length 2.2 kb. But I didn't get the band. So I split it into two halves. When I try to amplify it, sometimes I get band with non specific bands and sometimes I got no bands.
I did gradient PCR and I got band in one specific temperature. But when I tried to amplify the samples I am facing the problem. I used different types of polymerases but am not getting the specific band, please help me to get rid of this problem
yes, please tell us your PCR conditions. This would help us to think about an answer. Another recommendation from my side would be to use PCR additive (in case your DNA template is GC rich). For example BSA (Bovine Serum Albumin) or DMSO (Dimethyl sulfoxide).
BSA increases PCR yields from low purity templates. It also prevents adhesion of enzymes to the reaction tubes and tip surfaces.
DMSO is thought to reduce secondary structure and is particularly useful for GC rich templates.
did you try to increase the cycles for example 40 or 45 cycles, also your annealing time should be at least 80 sec. and annealing temperature maybe less than tm or your primers by 5 degrees
Chaitnya your gene of interest is not that long which need to be split...normal polymerase can also work for that... I thin the porblem is in pcr conditions or GC content...It may also be the problem with your thermocycler (PCR machine).. frst try to do the reaction in some other machine.. and as rightly suggested by Anna use some additives such as DMSO which break the secondary structure n hence the unspecified amplification. All the best.. and plzz feel free to ask more...
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India