Hello all,
I am in the process of writing a lab report about two lab exercises we just had at uni. The first one was a PCR of a DNA fragment, containing one of three variations of the CFTR gene, which went perfectly fine and the results from the gel electrophoresis were consistent with the expectations.
In the second lab exercise, we were tasked with determining the variation of the gene we were given through a RFLP analysis. To do that, we cut our samples from the PCR with two different restriction enzymes. My two samples are in the top right corner, beside the DNA ladder and uncut control. Since the samples were never cut, I got the mutation which removes the restriction site. My problem is that the two samples come from the same PCR product and should have the same length, but one appears to have run shorter than the other. I noticed that the DNA ladder on my side of the gel has run to one side, meaning that there was probably something wrong with the gel itself, rather than my samples. Furthermore, the uncut control (left of the top right DNA ladder) also appears to be smaller than my samples which is another throw-off for me.
Does anyone know why this could have possibly happened? How could the method be improved in that aspect?
I appreciate the help!
Hey Yoana,
my guess based on your picture without further information about the restriction enzymes and restriction sites (and corresponding restriction products) is that there is no difference in size between your two samples and the control sample. It's just an effect of uneven migration.
I've annotated your picture below and sketched out the wavy/bowed/arched pattern in what samples seem to travel. Clear size difference is of course in samples 5, 6, and 11. Samples 2, 3, and 4 might be double bands, would require to run the gel for longer to see if they separate. Your samples 12 and 13 look just like the control 14 or the other samples 7–10.
The rest of the difference you can observe would by my guess account to uneven migration resulting from imperfect electrophoresis conditions. Maybe the agarose is uneven depth, maybe different amount of salts in the samples, maybe uneven pH, crooked table, etc...
Thank you so much for the assistance, David Novak ! This was really helpful!
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