I'm trying to extract gDNA from C. elegans for sequencing using the following methods:
- Lysis using lysis buffer activated with proteinase K
- Freeze for >1hour at -80°C
- Heat for 3 hours at 60°C
- Heat for 30 mins at 90°C
- Followed by gDNA purification using AMPure XP beads
When I run the purified DNA on a gel (see photo - two lanes on far right), I come out with a strong band at 100-200bp, with little to no smearing of the whole lane (suggesting it isn't random fragmentation but fragmentation of a characteristic length). I understand that some fragmentation is unavoidable but why are my fragments so short and regular?
I agree with Tom Masi answer that most likely it is degraded RNA. But also realize that it could be degraded DNA. If degradation is sufficient you won't see the typical smear from high MW to low MW but it can nearly all be converted to low MW species.
Thanks both. I added an RNase step and the gels came out with high molecular weight DNA so it must have been degraded RNA.
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