Question

Day 1, You have isolated DNA from 30 individuals and performed restriction enzyme digestion. You have loaded the product and run the gel electrophoresis following the instruction properly. You expected to see DNA bands after the procedure is completed, like figure A, but after the completion of the run your gel looks like figure B. You did not see any DNA band. You were so upset, but your lab partner fixed the problem and your gel looked like figure A. What did he/she do?

Day 2, You repeated the experiment. You know that you have loaded the DNA samples and ran it for 30 minutes as instructed and observed the gel, as you should. But you don’t see any DNA band. You asked your friend to help. He/she told you that you will need to cast a new gel and run the samples again. What might have gone wrong this time?

Answer 1

DAY 1 : ​​​​​​

We cannot simply observe the DNA bands. That's why EtBr is added to the casting gel. EtBr is a intercalating agent which is generally used as a fluorescent tag. EtBr binds with the DNA and make the DNA bands visible under UV light. So, after completion of running the gel, the gel should be observed under the UV transilluminator with UV spectra. Only then the gel will look like figure A.

DAY 2 :

There may be a few reasons due to which you didn't see any DNA band on the second day. First of all, you have to add bromophenol blue or other such gel loading dye in the sample. It will help you to monitor the progression of the band through the gel. Stop the process before the band reaches near the other end of the gel. Secondly, remember to add EtBr during casting the gel. It will help you to visualise DNA bands under UV transilluminator. If you did all these steps correctly then the problem lies anywhere else. During connecting the plugs of power supply unit with electrophoretic chamber, put it in your mind that DNA is negatively charged, so it will migrate towards anode. So, you have to put the casted gel in such a way so that the wells would be placed near to the cathode, means opposite to anode. If you place it in wrong direction, the sample DNA will run out of the gel in opposite direction. And maintain the voltage of the power supply unit correctly. Voltage depends on the time of sample you're running and the thickness and fragility of the gel. Higher voltage can break the gel apart, whereas lower voltage can results in not moving the DNA out of the gel. And an another thing, always maintain the concentration of agarose in the gel according to the type of sample. Concentration will vary for plasmid DNA and genomic DNA according to their molecular size. Low concentration of gel can cause the DNA to run out of the gel before the process ends.

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