I am using degenerate primers with cDNA as the template.
Here are the components I used.
[ ] final
Buffer S 1X
MgCl2 1.75mM
DNTPs 0.2
Forward 0.75uM
Reverse 0.75uM
PfuPol 5U
Annealing temp is 60
Im gonna try to lower the PfuPol concentration
Hello, you are getting smears for two reasons. As it is obvious through your gel your gel and samples preparation needs more improvement: You need to be more careful especially while preparing your samples and take all the time you need because you are dealing with cDNA which is a stable materiel compared with RNA. The second reason you are getting the smears is the specificity of your primers or of your program (for example, the temperature of hybridization...), so you need to check again all the these parameters related to your PCR: the different temperatures, number of cycles...they are more important for a good detection. Good luck .
Hi Iskander I appreciate your response.
I used Goodview stain on the first one and Gel Red on the second.
Yeah I think I still need to figure out the optimized profile and components.
Thanks.
Hi Mahshid I appreciate your response as well.
I used 35 cycles with 2 minute extension time.
Alright I'll try lowering the template concentration.
I'm still hopeful that my primers would work. Haha
Thanks
Hi.
I think the degeneracy of your degenerate primers is very high (>1,000?). The higher the dengeneracy, the more the unrelated PCR products. If the degeneracy is too high, try to re-design the primers.
If the sequences can not be changed, try to reduce the concentration of MgCl2 (1.0-1.5mM). Also, you can change the annealing temp. as well as annealing time of PCR. In my experience, increase the annealing temp results in less PCR bands generally. If you have a gradient PCR machine, it will save much time.
Hi Ramina,
I hope things are getting better but, if not, also try checking the basics (autoclaved water, DNase free things, running the gel at a V that doesn't increase the temperature so much...), I know this might be obvious but if you see your second picture, there's also a smear in your DNA ladder (length markers) so maybe this Is contributing to the end result too.
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