I am currently working on Candida species, with a particular focus on Candida utilis, and I am facing challenges with my RAPD PCR experiments. Despite following a protocol similar to those described in the literature, I am not obtaining any bands in my PCR results. I am looking for advice on how to optimize my approach and would appreciate any suggestions or insights from researchers who have worked with RAPD PCR on Candida spp.
One common reason for a primer not working is that the annealing temperature is too high. If your primer is old and stored in water then take a new aliquot and store it in TE then try an annealing temperature gradient of 10c below the calculated TA to the Ta
I recently ordered a new primer and prepared its dilution. The annealing temperature mentioned in the paper is 36°C. However, when I applied a gradient PCR with temperatures ranging from 33°C to 40°C, I did not observe any amplification at any of the tested temperatures.
My primee sequences
OPA07-GAAACGGGTG
OPA09-GGGTAACGCC
M13-AGGGTGGCGGTTCT
yea the Ta are 39, 42 and 59 so if your reagents etc are working you should be getting bands so I would check the od260/280 ratio of the dna and choose a dna with an od260/280 close to a value of 1.8. If you have another ordinary pcr primer set then as a control run that primer set on your dna to check that the reagents and pcr machine are working correctly even if the Ta is too low for the proper pcr amplification. Also look for a diffuse low molecular weight fuzzy band of less than 100 in case the primer is self dimering and if it is then use a hot start polymerase
yes, I am getting a DNA concentration of 1.8 to 2.0. I have also performed the PCR using a universal primer set to check the PCR machine and reagents and have gotten the results. However, I am facing issues only with RAPD PCR. The setting I use for this PCR is:
94 for 3min
94 for 1 min
33-40 for 1min
72 for 2 min
72 for 7min
done for 35 cycles.
Good . cycling parameters look safe and should work. I think that this leaves 2 possibilities.
1 the primer has degraded.....try a fresh aliquot from the stock solution and
2 the dna has pcr inhibitors. run a pcr with 100ng, 50ng, 25ng, 12 ng, 6 ng and 3 ng. Sometimes less dna means less inhibitor and the pcr works better with less dna.
note an od260/280 ratio above 1.8 usually means rna or some salts from the dna purification kit is present in the dna. what is your dna source...plant,human etc and purification method? and how much are you using in your pcr reaction
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