Agarose gel electrophoresis was run on samples of cDNA obtained from RT-PCR with specific primers, and scans of gel were analysed in Biorad.
I'm having trouble ascertaining whether the molecular weight estimated in the program is comparable to the desired molecular weight of the genes whose expression I'm analysing.
For eg. gene X is 511 bp, bands observed from two samples in gel is approx. 484 bp and 521 bp (when compared to MW ladder)
Can these values be comparable to the desired 511 bp and thus represent gene X or not?.
I understand they are observed as not the same molecular weight but to what degree can I chalk that up to how well the gel was loaded and run.
DNA ladders give approximate results. The speed of migration of dna in agarose depends on the surface charge of the dna and that dependes on the base composition. So The ladder may be AT rich and although the band is 500bp on the ladder you will find that a GC rich 500bp amplimer will run at a different apparent size, Conformation also brings different bases to the surface of the dna molecule and this will also change the speed through the gel. Plasmid dna bands show how much the migration of dna can change with the confirmation. I think that your band sizes are close enough to the expected size to be the right pcr product but you can always sequence it for exact confirmation
When estimating the molecular weight of DNA bands on an agarose gel, there are several factors that can affect the accuracy of the estimation, including:
Given these factors, it's difficult to say with certainty whether the bands observed on the gel are representative of the desired 511 bp fragment. However, the fact that the observed bands are within 10-20 bp of the expected size suggests that they may be representative of the gene of interest. It's also possible that the differences in size could be due to experimental error, such as slight variations in loading or migration. If accuracy is critical, it may be worth repeating the experiment and optimizing the gel conditions to improve resolution and accuracy.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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