I've been performing RAPD-PCR for the yeast cultures I isolated from the Sauerkraut and everything was great. Subsequently I started to perform RAPD-PCR for the bacterial cultures I got from the very same source. Unfortunately I wasn't that lucky this time. When I carried out electrophoresis, the first few gels I received were just fine, with all the bands right there where they are ment to be. But this is when the the troubles began - I tried to perform electrophoresis for the next bacterial isolates (same conditions - 100V for 1 hour). At first it was also okay, I got the bands, but during one of the following electrophoresis I got no bands at all (only the marker). During next analyses everything became blurry. I modified the voltage (70V and 85V) but everything remained the same. I even tried to alter the time a bit. The buffer I use is 1xTAE. Can it be that during one electrophoresis (when I didn't see any bands - only the marker), the voltage and time were inappropriate so that the DNA migrated into the buffer, so now I'd have to change it? I replaced the buffer regularly, but now I'm just not sure what can be the reason.
I'll really appreciate all the help I can get here. Thank you in advance!
why do you think the electrophoresis is at fault. Are you running a positive control that usually works in your experiments. Could it be that the pcr did not work due to primer degradation or some similar problem. If your size standard is working then it seems that the pcr is not working. I would use new regents particularly the primer and definitely if your primers are made up in water not TE
Hi Szymon,
Check the following link on some tips and tricks to improve agarose gel electrophoresis.
https://www.lifetechnologies.com/za/en/home/life-science/pcr/elevate-pcr-research/agarose-content-with-tips-and-tricks.html
why do you think the electrophoresis is at fault. Are you running a positive control that usually works in your experiments. Could it be that the pcr did not work due to primer degradation or some similar problem. If your size standard is working then it seems that the pcr is not working. I would use new regents particularly the primer and definitely if your primers are made up in water not TE
also the template should be checked, I also think it is the reaction problem, not the electrophoresis.
I observed a similar situation recently. If you are able to see the marker, I don't think that electrophoresis is the problem. Probably something went wrong with the PCR, but in our case there was absolutely no reason and after testing every possible variant, it worked again with the same conditions as the very beginning. I think it's better if you check your PCR or keep trying as we did.
There is no two way about electrophoresis most especially agarose, if all reaction conditions are kept constant and amplification occurred then check the temperature of the buffer during electrophoretic mobility. I carried out gene expression on the amplification of oestrogen receptor gene after dosing, all conditions kept contact and aseptically but realised that i could not see any band, i repeatd this over and over again until i noticed that the buffer is always cooking the gel, the temperature had gone up that i cant image. so pls take note of the buffer temperature
:You are doing RAPD with 10mers as primers. There will always be inconsistency in bands but not totally disappearing. This could be mainly due to your template DNA and other components in the PCR reaction. Sine the markers are OK the problem cannot be in agarose electrophoresis. So please check the template DNA quality, primers and the other components.
Guess your DNA template is the issue and not electrophoresis. Purify your template using silica columns (any PCR/gel band purification kit will help) and then try repeating the PCR. If this does not solve, then add some additives (some detergents, glycerol etc. or may be commercial ones) and will definitely help. For consistency in PCR profiles, use extremely carefully quantified and equal quantity of DNA template and make a PCR master-mix before hand for all the samples which you want to compare. If you have custom synthesized primers, be extremely careful for dissolving them in same manner for every dilution. Only add the Taq just before setting PCR. Technique per-se cannot be blamed for less reproducibility but proper setting and controls are a must. Before you begin comparative profiling, define a specific sample as your positive control which you will include in every PCR/gel to size each amplified band. This will help you to standardize each primer PCR profile.
I've been trying to deal with my DNA template over and over again, thus I thought failures may be a reason of PCR. However, I shall try once again as soon as I get back to laboratory - I wanted to ensure whether these failures may be a cause of PCR. Thank you all once again!
check your DNA and PCR reagents .Your case has nothing to do with agrose elecrrophoresis
It turned out that the kit I used was to blame. I succeeded in obtaining the DNA with the kit I used in the first place. Obtained DNA was pretty clean, as well as the amount of the isolated material allowed to proceed with the tests. Thank you all one more time!
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