Hi,
I am currently working on ISSR primers for my algal samples. When I did PCR for my samples along with some marine algal specimens, I could observe bands for the marine specimens and a smear for mine. Why is that? What does this mean? The purity of DNA was measured using nanodrop and it ranged from 1.7-1.9. Kindly suggest on how to proceed to get clear bands for my samples.
DNA isolatin method - CTAB-4%
PCR reaction - 5.5ul master mix, 15.5ul water, 2ul primer and 2ul template DNA
PCR conditions - Initial denaturation - 94 for 7min,
Denaturation - 94 for 30 sec
Annealing temp - 45 for 1 min
Elongation - 72 for 30 sec
Final extension - 72 for 7 min
The rough image of the gel is attached for reference. The first gel corresponds to the marine algal specimen and the second gel to my specimen of interest.
Thanks in advance
Swetha
Absence of bands or unclear bands could be due to degraded DNA or the PCR condition is not fully optimized. For bands smearing, it could be caused by non specific products or again, caused by non-optimized PCR conditions. So u can try to change the PCR conditions like adjusting the Tm etc.. always run optimization at different conditions and see at which conditions u can get the most intense bands. Good luck!
I agree with Zx Chong, in addition you can try PCR with other set of ISSR primers - it is often the cheapest and fastest way to get better results (PCR master mixes are generally more expensive than primers)
If your template is quite big, you can try increasing your last 72°C step to 10 min instead of 7. Also, you could try running your gel a bit slower, 7 volts/cm of gel is the recommended voltage, I think.
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