I need your help with optimazing my PCR reaction. My PCR product should be 850bp. Starters Tm is 65, they do not form dimers or secondary structures and they’re specific (Blast doesn’t show any unspecific product that may occur). I've tried gradient PCR with typical mix and with Hot start polymerase and every time I receive strong band that is 150bp. What can I do to get rid of this unspecific product and what can it be?
Here missing important information. What is target sequence? Do you have positive control? Is your DNA from pure culture or mixture?
Hi Moein,
As your desired amplicon length is 850bp but you got amplicon at 150bp, it's a clear case of non-specific amplification!
Now the question is where or which segment will it amplify as you got no other gene while doing primer-blast !!!
Well, the answer is also here...Look, as your Tm is 65 (very much higher) obvoiously your primer has high GC content! So the primer may bind to the CPG island or any other GC-rich region, which would never be shown in the BLAST result ! It's better to redesign your primer with the desired features including GC content,
Hi, something similar happened to me, I was not able to get the desired product using Taq polymerase. However, when I used SYBR Green PCR Master Mix for PCR I was able to obtain the full-sized product. (The product was utilized as IVT templates so the presence of dye didn't affect my further processing).
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes