I am trying to design MSP primers for human BCL11A gene based on the DNA sequences from the CpG Islands - 115 & 106 prior the promoter region and 105 towards the end of the gene.
I have attached the Excel file with all the primer sequences for these regions. The graphic representation from the result aligned the primer with input sequences and I double checked the parameters of the primers. However, I couldn't see any bands after running the PCR. The PCR conditions that I used are:
Mixture: 1microl primer, 10microl master mix, 7microl deionised water and 2microl genomic DNA
5 mins at 95oC
30sec at 95oC
30sec at 55oC (chosen based on the primer pair with the lowest average Tm)
30sec at 68oC
(Repeat step 2-4 for 40 cycles)
Another question I have is when you are preparing for PCR, do you mix both unmethylated and methylated primer pairs in the same sample mixture but separately?
I am clueless why the PCR's not working. Please help!!!!!!! And thanks a bunch!