I am trying to amplify a gene using genomic DNA and Taq polymerase. I got two clear bands, one is the specific band with very low intensity and another band which is non-specific with very high intensity. I used Gradient, in that more the annealing temperature , lesser the band intensity (both bands).
Genomic DNA- 50ng
10X Buffer with 20mM MgCl2- 2.5 μl
dNTPs- 0.5μl ( 200μM each)
50mM MgCl2- 0.25μl
10pmol/μl primers- 1μl
Taq polymerase-0.25μl
Total reaction volume(25μl) adjusted with MQ.
Conditions:
Initial denaturation- 95°C for 3 min
Denaturation-95°C for 30sec
Annealing - 53-56°C for 30 sec
Extension - 72°C (1 min/ kb)
Final extension- 72°C for 7 min
Hold @ 4°C
Number of cycles-35
How to get rid of non- specific band and how to get a specific band with good intensity? You
can you mention the size of your gene of interest and the non specific band?
I think that the Mg concentration may be too high causing the non specific band and with all long pcr T taq often works well at 1.5 mM Mg) it is best to have high annealing temperature primers .Make the primers longer so you can have a cycle annealing temperature of 60c or higher
First if your 10x buffer contains magnesium chloride, you don't need to supply it separately. Excess magnesium chloride don't increase the activity of taq polymerase. You can use di methyl sulfoxide to facilitate the annealing of your primer. This will reduce the probability of creating non specific band. Also do a primer blast using your primers in NCBI website. Through this, you will be able to know whether there are any other sites in the genome which will bind your specified primer.
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