when I run my PCR reaction I got two product one sharp non specific product ( 300 bp) in addition to the target product which appear very faint 461 bp.
the get photo is attached
1. What is the PCR conditions? So we could have an idea what's the problem, also include: the Tm of you primer set.
2. Please do gradient PCR that will optimise your Ta.
3. There could be two problems PCR conditions or the primer itself.
Thanks Richard for your answer
PCR condition
94 c for 5 min then 30 cycles of (1) 94 c for 40 sec (2) 56 c for 35 sec (3) 72 c for 40 sec, then 72 c for 7 min
I tried 3 annealing temp 55, 56, 57 c. and I got the same results
Tm of forward primer is 54 and for the reverse 56
I think you have to increase the annealing temperature in order to eliminate the nonspecific binding, may be over 60c. I got the same problem here in my research.
1. I agree with Fawaz but also try to reduce annealing time to reduce the opportunity for other non-specific to anneal also to reduce the elongation time to reduce the opportunity to elongate the non-specific binding.
Initial: 94C for 5 min
Den:94 for 40 sec
Ann:60 for 15 sec
Elo: 72 for 20 sec
FInal Elo: You can remove this step. (necessary if PCR product is >1000 bp)
2. Also for in silico checking, try to BLAST search the primer set you have so you could check if there are truly non-specific targets (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).
3. Try to use HiFi (high fidelity TAQ) if you have some.
4. "One day it work, tomorrow it might not" - my usual happening in the lab.. Good luck so be patient.
Good idea is check the primers if you take its from literature.
For increase the quantity of the target you must perform gradient of temperature, cycles and eventually Mg2+.
Good luck
Hi Rawan,
I would probably add some PCR additives to enhance its specificity and increase its yield. I usually use 2-5% 'DMSO'. There are other additives you can use.
See this site for different PCR additives: http://www.staff.uni-mainz.de/lieb/additiva.html
It is also possible that you are amplifying a GC-rich template. GC-rich template is usually hard for PCR amplification. Please see the attached document for solutions, which I obtained from below website:
http://bitesizebio.com/24002/problems-amplifying-gc-rich-regions-problem-solved/
Some PCR kits might already contain reaction buffers which are ready for GC-rich template amplification. Check yours.
- Badges
- Science topic