Hi,
I recently ran a PCR Reaction using Taq 2x Master Mix RED - Ampliqon
The primers I used were both 10µM.
I added 2µl same template human cDNA
and diluted to 10µl total with water.
The cycle used was as follows:
- 95c | 5'
- 95c | 30"
- 60c _ 61c _ 62c | 30"
- 72c | 1'
- 72c | 10'
I ran the products on 1%agarose gel ,100v and for 1h.
all the product were approximately 900bp and as you can see, showed nonspecific bands.
how to troubleshoot ,
and how will the problem be resolved?
Any suggestion or comment in this regard will be helpful
Thanks
niloufar
you can increase the annealing temperature (gradient if possible) and with increasing temperature the pcr will become more specific. Alternatively use a working temperature and add amounts up to 6%dmso and this may cause the primers to anneal more specifically and give a better and cleaner result
What is your target gene?
Do you have positive control?
What is GC% of your primers?
Did you blast them?
@alirezamordadi
We are working on CD20 gene.
-No
-they are standard
-yes
Niloufar Mohammadkhani
In my knowledge for CD20 gene studies use fragment of gene.
So if you have i.e primers f-r, f-r1 and f-r2 your bands seems right and according to dear Paul Rutland use gradient PCR.
Good luck
I agree with paul Rutland with primer annealing temp.
your samples looked not clean and there was degerdation.check the quality of DNA
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