Background: I conducted PCR on several grids plate isolations to amplify the DNA for gel electrophoresis. Before running the gel I noticed sediment, or precipitant in my PCR tubes. I thought that I needed to resuspend the DNA. I vortexed 4 of 5 samples, in ascending order. From well sample 5 to sample 2, sample 1 was not vortexed. I was made aware that vortexing wasn't recommended so I stopped. I did try to pluck and aspirate sample 1.
I used a ladder and filled 5 wells of the gel. The gel ran for 13 minutes and I got alot of migration, and smearing in wells 3-5. The migration in wells 3-5 exceeded the ladder. I sheared my DNA. I didn't observe any banding or migration in well 1.
Questions:
Yes, based on your description and the gel image, it does appear that the DNA may have been sheared. Smearing usually suggests degraded or sheared DNA, which can occur due to excessive handling, such as vortexing. Vortexing can shear long DNA molecules, leading to smaller fragments that create a smeared appearance on the gel. Since you vortexed samples 2 to 5, this likely caused the observed smearing and excessive migration, especially if the migration exceeded the ladder, indicating fragments of varying sizes.
The absence (actually this is not true - there is a band) of band or migration in well 1 might be due to another issue, like insufficient DNA loading.
Avoid vortexing the PCR product and instead, use gentle pipetting to mix.
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