I got this band pattern on 1 % agarose gel after doing one step RT PCR for a particular gene. I was expecting a lot of sharp and clear bands. What can be reason for getting such a diffused band?
Hi, for me it's not possible to see a picture. Only my problem? Cheers, Nadine
Hi Prikanka, could you upload your gel photo, I am not able to see the photograph. Have you checked the quantity and quality of your extracted RNA? Very often, if the quality of isolated RNA is not good, one cannot expect a good quality cDNA, which, i think, you would use it as template for your specific gene target... very often errors start with the RNA isolation
But also the ladder ran not properly. Change the buffer in the electrophoresis chamber and repeat your experiment.
Hi again, seems the ladder has not separated well, perhaps if you ran the gel longer, you would be have to a clear-cut observation... Yes I also agree with Nadine's answer.
Second, I think that those smears of your PCR amplicon do not give definite bands.
In my experience (not that you asked :) but just to give you heads up) , that happens when the quality total RNA extraction is not good. I would suggest: first check the quality of RNA. Only if you are satisfied with the quality, then start cDNA synthesis. Here I have inserted a picture of the total RNA I could finally get (after many2 standardization, I used TRIZOL for RNA extraction). I have also inserted the PCR amplification after I could standardize good quality RNA extraction...
Heads up!! keep going, you will get there.. Cheers!!
Hi, you have to increase your agarose concentration to 1.8 or 2 %, and also check RNA concentration before RT-PCR like tell you Rita and I also agree with Nadine s answer
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