Hello
I have done PCR using a 18S rDNA universal primer and eluted the PCR product from the agarose gel using RBC kit. Eluted PCR product on agarose gels is showing multiple bands on gel electrophoresis.
The PCR product of mine only showed a single band but after elution I am getting multiple bands...why am getting these multiple bands after elution?
Hi,
I think you have degraded a PCR product after elution. have you respect the protocol?
You should get the same band if you eluted it from the gel. Did you run both agarose gels at the same conditions? did you see your band of interest on the gel you run after elution ? I think there are something in the kit that had caused it. is your elution buffer free of DNAse or restriction enzyme ? In that case try to do it with another kit and see what happens. However if you go the multiple bands from the eluted PCR product it's understandable.
1.check your gel extraction kit and buffers to see whether are contaminated or not especially elution buffer...
2. in eluted DNA gel electrophoresis can you see your desired bands beside unwanted bands? if yes it means that some how there is a DNA contamination, if no it means that some how your DNA has been degraded again contamination but this time DNase or maybe not gently handling...
3. are those multiple bands sharp enough to judge them as a real band? (in this case you must have your desired bands besides.)
Thank you very much for your reply...
I have eluted DNA by two gel extraction kit (RBC and Quiagen) but getting same multiple band...the desired band I obtained but I always get another unwanted band is of double length (desire band length 2kb, i got another 4kb band length) but unwanted band is faint not so sharp.
so you get TWO bands... one is unwanted and it happened for SEVERAL times... and it is always DOUBLED size (bigger than desired band) and faint... and if contamination possibility is eliminated.....have you tried to have another gel extraction for the previously eluted fragment???? maybe you will face the unwanted band again ... I dont know exactly what is going on at molecular level for your DNA but... depending on your goal, if you want to clone your gene in a vector would suggest you to carry on with this faint band you will get your desired colon.
after PCR you had a single band after elution you have two bands. In this case it is not contaminated and you can continue your work. Why two band after elution I believe that chemical interaction (you should see a chemist). I'm sorry
Prabhakar, You should repeat your experiment with carefully as per genuine suggestions of researchers. I hope you will get the results
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