I am currently looking at the expression of the Gene POU3f4 in C2C12 cells during different differentiation stages day 0 to day 8.
When I use cDNA i got many non specific bands in contrast if i Use gDNA I got clear bands with expect product size.
I have tried to decrease Mgcl concentration without any benefit.
I think that pou3f4 only has one exon so alternative splicing does not seem very likely so I assume that there are similar genes and you are getting non specific priming. Your choice is to redesign the primers with primer blast or running a temperature gradient of annealing temperatures. A similar approach is to run a gradient of DMSO to see if most of the bands disappear at either a higher annealing temperature or at a concentration of DMSO somewhere between 0 and 10%
Yep, my guess is non-specific products from related genes. I'd try gradient PCR to find a better primer annealing temperature and/or designing new primers.
Thanks for the above answers. I have tried about 9 pairs now without any success. I have established the best annealing temperature ranged from 63-60 C. I am not sure why I got clear bands with gDNA though?
the reason gdna does not usually work is that there is usually an intron between the primers but with a one exon gene both the cdna and gdna contain the same sequence between the primers
- Badges
- Science method