Hello everyone.
Please help me to understand this agarose gel. I did a plasmid (Pgem vector with an insert with an expected size of about 3kb, from E.coli) extraction with a simple protocol Birnboim e Doly (1979). The first two bands are the different topologies of the plasmid. What I need to know is the last band (very intense) - It's very small and lower than 100bp (maker is 100bp plus). What could it be? Thanks
It looks like degraded RNA, as RNA is not stable in alkaline pH. Simple and short RNAse treatment will remove this contamination.
I concur with the above that the bright spot at the bottom is likely to be RNA. But I would also point out that your mini prep did not work all that well, pGEM plasmid mini preps should give you a lot of plasmid DNA but you have mostly genomic DNA and RNA. I would certainly give your mini preps another try.
Two likely causes:
1. You got RNA because you forgot to add your RNAse or your RNAse is very old -
plasmid extraction kits do not differentiate between DNA and RNA, but use RNAse to remove RNA.
2. You got genomic DNA because
Continuing on my previous answer (the ResearchGate app has some issues) - You got genomic DNA because you did the cell lysis for much too long or you vortexed the cells in the lysis buffer. The kits seperate genomic DNA from plasmid DNA by size - long DNA percipitate with cell debree
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