I have been trying to assess molecular diversity of rice plants using ISSR primers. I used CTAB method for DNA isolation. I have tried with different reaction mixture conditions and PCR amplification conditions. Still I get weak bands. Can anyone suggest me with a standard protocol?
Have you quantify your DNA samples??
Use around 30-50 ng of DNA for proper amplification. Check primer concentration. Recheck PCR protocol, specially annealing temperature.
If possible go for PAGE instead of agarose gel electrophoresis.
All the best....
Hi:)
I sugest to use termal gradient during PCR to find optimal annaling temperature.
adding 5 cycles extra also could be helpful
Hi,
I also feel that first you check for the quality and quantity of you DNA at 260/280nm
Secondly I would like to know what is anneling tempertaure you are using.
I have done ISSR analysis of bamboo (Poaceae) and followed this cycle.
Initial denaturation at 94ºC for 5min followed by 34 cycles of denaturation at 94ºC for 45s, Primer annealing at 50-52ºC (depending upon the primer) for 1min., Primer extension at 72ºC for 1min with the final extension at 72ºC for 10 min.
Hope this will help you.
All d best..
Hi Dear,
Recently we have done molecular diversity analysis of rice using SSR markers. You can get sufficient DNA by using CTAB method. But please check DNA concentration if it is sufficient around 20-25 ng/ul. Please check concentration of stainer and staining time
PCR has performed in 10 μl reactions containing around 25 ng of DNA template (3 μl DNA with 10X dilution factor) using GStorm thermal cycler. Ten micro liter of mineral oil was added in each well to prevent evaporation and the PCR plate was wrapped with adhesive film.
The ingredients of each PCR reaction for SSR markers are as follows:
H2O = 3.35 μl
PCR buffer (10X) = 1.0 μl
MgCl2 (25mM) = 1.35 μl
dNTPs (10 mM) = 0.2 μl
Primer F (10 μM) = 0.5 μl
Primer R (10 μM) = 0.5 μl
Taq (5 U/μl) = 0.1 μl
DNA (25 ng/μl) = 3.0 μl
Mineral oil = 10 μl
After initial denaturation for 5 min at 94°C, each cycle comprises 45 sec denaturation at 94°C, 45 sec annealing at 55°C, and 1:30 min extension at 72°C with a final extension for 7 min at 72°C at the end of 35 cycles.
Wishing you get solved of your problem soon.
Thank you all for your valuable suggestions. Mr. Arvind Kumar Goyal, i would like to know the primers used for your research
These were the primers used in my study:
(GA)8T
(CT)8G
(CA)8G
(TC)8A
(TC)8G
(AC)8T
(GA)8YC
(AC)8YA
(GACA)4
Hope this will help you.
Dear Mr. Arvind. Thank you for the information. I ll get back to you after my experimentation for further clarifications
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