Genomic DNA isolation

I am isolating genomic DNA from C. elegans and have noticed some differences between protocols I’ve found online and what I’ve done in the past. Several protocols mention using agarose instead of agar for the NGM plates because of problems with contaminants in agar. What kind of problems tend to occur? I’ve always used agar in my NGM, so what should I look for to make sure it hasn’t caused a problem? I usually wash the worms off the plate using M9. Is there an advantage to using the sucrose method? I have also noticed the recommendation to incubate the worms in 0.1M NaCl for an hour to help digest bacteria in the worm gut. I have never done this either. Any thoughts on how important any of these steps are for genomic isolation of DNA? I will be using the DNA for PCR.
Ellen Tarr

Don’t worry about the NGM.
M9 basically contains 0.1M NaCl. 0.1M NaCl is faster and cheaper to make and can be used for washing off worms and incubating them for a while. For longer periods better use M9.
For transcriptomics or proteomics studies you should be more careful with allowing the worms to digest gut bacteria, for your PCRs it doesn’t matter if there are some bacteria around. Btw, for rapidly testing strains you can also run a PCR with one single worm per PCR tube.

For people doing genome resequencing, how stringently is it necessary to remove E. coli when preparing DNA? Has the proportion of E. coli reads in the dataset been a problem e.g. significant reduction in C. elegans coverage?

I resequenced 4 genotypes this year using mixed stage animals thoroughly washed in M9 (>=6x) and found in all cases ~95% of my reads that passed quality filtering were alignable to the C. elegans reference genome using MAQ.

has anyone done genome sequencing on embryo populations from bleached gravid adults?

Regarding Ellen’s question about agar vs. agarose, one difference we’ve found is when quantifying the DNA concentration by spectroscopy. Some contaminant (oligosaccharide?) from the agar plates absorbs UV, so the measurements are artificially high. We use gel electrophoresis and ethidium bromide staining to estimate the actual DNA concentration. Other dye-binding methods should also work. Note that the contaminant co-purifies on Qiagen columns and also is precipitated by ethanol. We have also observed a similar contaminant with some batches of agarose, although the amount is much lower.

Good luck,