Hi, I'm trying to develop a KO cell line from an established cancer cell line. My gene of interest is present in 3 copies in this cell line.
I'm using a multi-sgRNAs technique to increase my chances of a significant deletion. I isolated 4 clones of interest which share a similar trait: they all show 3 different bands after PCR amplification an electrophoresis on agarose gel. This is not so much a concern, since it was one of the expected outcome (the CRISPR/Cas9 system can create three different cutting pattern, resulting in 3 different bands). FYI, the 3 bands are all different in size from the WT band (with the top band being around 100 bp bigger than the bottom lane).
I ran again the sample on a more concentrated agarose gel (2%) with a lower voltage to get nice bands and being able to cut them. I extracted DNA from each band and re-run a PCR on each of them to increase my DNA material. For all my 4 clones, the bottom band amplify to a nice and single band corresponding in size. However, the middle and top lane display the 3 bands again, and it doesn't make sense to me. Indeed, I could understand finding the middle band in the top band sample, or the other way around. But I would have never expected finding the bottom band in the top band sample, because the top and bottom band are clearly separated and shouldn't contaminate each other.
I made a mistake by not using sterile instruments to excise my bands, which could explain in part some contamination. However, if it was this issue, I should have multiple bands in the bottoms sample, which I don't have, and I should have cross-contamination through all the sample, which is not the case. I'm pretty lost so if someone has any idea, I would take the advice with gratitude.
(I attached the gel picture from where I extracted the bands (small gel) and the re-run PCR gel whit the unexplained bands. On the gel, T= Top band; M= Middle band; B= Bottom band).
Thank you all!!
Is it possible that your top band is a heteroduplex of the 2 smaller bands. The loop of ssDNA would slow the band down through the gel. If this is the case sequencing would be messy after the bubble in the heteroduplex or you could cut out the top band, heat to 96c and allow to cool slowly and if you find that running this denatured/renatured sample once again has 3 bands then the top band should be a heteroduplex
Thank you Paul Rutland for your answer, that's a really interesting point, and it could explain the situation for the top band. However, it doesn't explain the profile that I get with the middle lane. For example, clone 18, the middle and bottom band are well separated (as you can see on the small gel), but when I run the re-run PCR of the middle band, I get 3 bands as well. The presence of the top band could be explained by contamination/heteroduplex, but why the bottom band is also there? An heteroduplex situation can't happen in this case, that's why I'm confused.
As you say Romain Pacaud contamination is a strong possibility especially with clone18. The trailing peak of pcr product on all of the bands in the centre of each band looks like it is caused by an air bubble or unmelted agarose in that track and could be spreading small pcr product into the middle band. My feeling is that sequencing the bands from both ends will clarify what is happening. Other than that I cannot see a sensible scientific rationale but will be fascinated to know the answer when you solve it please
Unless ,of course, the small amplimer is created from both primers/one primer inverted annealing inside the middle product but only after deletion since the WT amplimer does not give this band then we do not need to invoke contamination
Paul Rutland Yes your last answer was brought up by one of my colleague, and that's a reasonable assumption, even if I feel it's unlikely because it would mean that this event happen for all the different clones, which is not impossible but with really low odds. Anyway your help was really precious and if I can get to the bottom of it, I will definitely share my conclusions with you! Thank you again for everything!
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