Dear experts,
I have done RAPD gradient PCR at different temperature by using takara pcr master mix without additional Taq DNA Polymerase and dNTPs. There is only one band upper ladder all the temperature (36-45 °C). RAPD primer synthesized from sigma.
Herewith I have attached gel image for your perusal.
How many base pair is you random primers? the length is 6-base pair? 8? 10? or 12?
if you use longer random primers, you may get few or even no band with viral genome in RAPD-PCR.
Hi!
First of all your PCR conditions are false.
Initial denaturation for 5 minutes is fine again you are denaturing for 5 minutes in cycling conditions also obviously your product/s would be denture and you do not get anything, that's what happening.
You need not do any gradient for RAPD, by the way who suggested! You should get many bands in RAPD.
Follow the mentioned
Initial denaturation 94C for 5 min
94C- 45 sec
35C- 1 min
72C- 2 min
35 cycles
then final extension 72C-5 min
load on 1.5% Agarose gel at high voltage so the band resolve clearly keep an eye on gel run so that bands will not run away.
It should work fine, try carefully. I am going out of station this evening and will not available till Monday. Then only I can answer your doubts.
Good Luck.
Refer all my answers about RAPD all your doubts will be cleared
Hi,
First, I agree with Jetty Ramadevi that the PCR conditions may false. I suggest using the following RAPD program that I used many many times and it works perfectly with all kinds of PCR machine brands and almost all DNA types whether from animal sources, plants or microbes.
94°C - 2min (1 cycle) Initial denaturation
92°C - 1min
36°C - 1min (40 cycles)
72°C - 2min
72°C - 10 min (1 cycle) final extension
I also suggest to adjust the reaction components as: 10pmol/µL of single primer, 25-50ng of genomic DNA.
Good Luck !!
As per Jetty Ramadevi suggestion
I got 4-5 bands from my two different RAPD primers by following PCR setup
PCR set up
Initial denaturation 94C for 5 min
94C- 45 sec
35C- 1 min
72C- 2 min
35 cycles
then final extension 72C-5 min
load on 1.5% Agarose gel
Respected experts,
I very thank you for given valuable suggestion, as per (Jetty Ramadevi) PCR set up with 2% agarose gel. i got good phage RAPD result,
Very good, lot of improvement. Keep it up.
No idea what series you are using, in general certain primers will not give any bands hardly one or two, omit that kind of primers during making trails. I suggest hot-start is always better for RAPD, I used to do hot-start when ever I use RAPD PCR. It is your wish whether to do or not.
Respected Jetty Ramadevi,
Thank you for your valuable suggestion, i need your help endover my research.
Nattan and everyone else I am sorry to come into this discussion a bit late. It is quite obvious that you had a problem with PCR conditions as pointed out by the respected Jetty Ramadev. I am just thinking loudly:- are your DNA samples stored in water or in TE Buffer. My experience with DNA samples stored in TE buffer is that they require to be diluted with equal volume of PCR water to give amplification results. The reason is that EDTA present in the TE buffer chelates magnesium ions in the master mix thus inhibiting the amplification process. Dilution with water reduces EDTA concentration, allowing good amplifications.
You are right Eliud.
In general TE buffer composition is 10mM Tris and 1mM EDTA. Especially for PCR reactions we make separate dilutions by reducing the EDTA concentration then absolutely no problem at all, try that it works fine. Either you can take 0.1mM or 0.01mM the lesser the better. But one thing even you can use the DNA dissolved in normal TE buffer and make dilutions either using distilled water or MilliQ water you hardly get any problem. Make limited dilutions store properly rather than making lump sum. Of course it depends on your work load in the sense how regularly use the samples for PCRs either microsat or ISSR, RAPD etc.
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