I am trying to run RAPD on my Lactobacillus sp. according to the protocol by Rossetti & Giraffa 2005, in Journal of microbiological methods. However i sample failed to show any band. Can anyone please help me on this? For the detailed procedure on how do i run the assay, I have transferred:
12.5ul of Tris HCl (Invitrogen, 10x PCR reaction buffer);
2 ul of m13 primer (5'-GAG GGT GGC GGT TCT-3'),
20 ul of sample DNA (unknown concentration, extracted by Bactozol kit);
3 ul of MgCl (Invitrogen, 50mM MgCl),
0.13 ul of polymerase (Invitrogen, Platinum DNA Taq polymerase);
1 ul of dNTP (Invitrogen, 10mM dNTP mix);
11.37 ul water.
Total reaction volume 50ul
PCR condition (Applied biosystems Thermocycler): 94oC, 2 min; Then 94oC 60s, 42oC for 20s, 72oC for 2 min at 40 cycles; finally 72oC for 10min.
With above condition I view the gel with 1.5% agarose in TAE buffer and stained with Sybr Safe stain (invitrogen). 1kb DNA ladder was used.
However, i failed to get any bands from this RAPD reaction, i have repeated the test for a few times and it remained failure. I am sure that i have extracted some DNA because i have used the same extracted DNA to run the 16S rRNA amplification and it was a success. I have attached the photo together with this question where: -ve means negative control, "a" is my sample "a" that undergone RAPD, "b" is sample "b" that undergone RAPD, "a 16s" is sample a that run through 16S rRNA amplification, "b 16s" is sample "b" that run through 16s rRNA amplification.
I hope someone can help me on this situation. Thank you very much.
have you try removing MgCl2 from the reaction? if not please try! also, forward and revert pair of primer, make sure you add them all, and how about reduce the amount of your primers to 1 ul, and reduce the amount of your DNA template or increase you Taq volume to 0.2 or more
Hi Dr Bongkot Noppon, thank you for your reply! I always thought that primers for RAPD does not need forward and reverse primers like other PCR reactions, for this, can you please help to suggest another primer for me? My literature only state 1 primer for this particular reaction. Besides that, may I know what is the reason behind in suggesting the removal of MgCl2? Thank you very much!
The first component in your reaction is the 10X buffer I think, but if you use 50µl reaction this should be 5µl of 10X buffer, not 12.5µl. You have the first line as "12.5ul of Tris HCl (Invitrogen, 10x PCR reaction buffer)" but I presume the Tris HCl is a mistake and it should be 10X buffer.
I suspect another problem is the DNA extraction. The 16S PCR is a very different sort of amplification reaction - it will work with much less DNA than RAPD amplification needs. You need to be able to measure the concentration of DNA from your extraction, then dilute to a fixed amount. Usually you would add 1 - 20 ng of bacterial DNA to each reaction, but that amount must be fixed for all RAPDs, e.g. add 10 ng to all reactions.
If you don't have a spectrophotometer you could estimate DNA concentration by comparisons with known amounts of DNA on agarose gels, but this is not best practice. A NanoDrop spec would be the best method, as you would only lose small volumes of DNA.
Hi, Dr Vincent Mulholland, thank you for your suggestions! I'll try to run the test again according to your suggestions. However, I have previously tried on estimating the DNA concentrations on the agarose gel, but nothing appeared. So I believe that your suspect on low DNA yield might be one of the reason for my failure as well. Thank you!
I've had same problem with RAPD lately. I'll also try doing it once again according to the suggestions Dr Vincent gave here and paying more attention to the things Dr Vincent mentioned here. I would be grateful, Ng, if you posted here how these suggestions worked for you. Best luck!
Hi, Szymon Strnad, I solved the problem by switching the entire protocol by using master mix reagents, which, the amount of sample DNA remained not standardized. Therefore, I am sorry that I still cannot figure out what is the exact problem from my previous experiment. Hope this help!
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