I recently moved to a different research institute and was happy to discover that my new lab had not one, but several different kits for yeast genomic DNA isolation. I like trying new kits and protocols, especially when they promise, like the Olympics, to yield results that are “Swifter, Higher, Stronger”. However, in this case I should have been alerted by the variety of kits, rather than encouraged by it – an array means that people are still looking.
I also should have been alerted by the phrase “take your bacterial cells…” in one of the kit protocols: it had clearly been copied from a bacterial manual. In the end, one of the kits failed to produce DNA of reasonable quality; the other resulted in usable DNA, but with a low concentration. My labmates have obtained similar results. I think I’ll stick to the tried and true DIY prep for a while.
This protocol was initially proposed as a method for recovering plasmids from yeast – you simply transform a couple of microliters of the resulting prep into E. coli – but it is now routinely used to isolate genomic DNA to use as a template for PCR.
1) Grow 10 ml yeast culture to saturation, preferably overnight in YPD at 30°C.
2) Collect cells by centrifugation, put tubes on ice. Decant supernatant, resuspend pellet in 0.5 ml of water. Transfer the cells to a 1.5 ml screw-top tube and collect cells by centrifugation.
3) Decant the supernatant and resuspend the cell pellet in the residual liquid by vortexing.
4) Add 0.3 ml of Solution C: 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8), 1mM EDTA.
5) Add 0.3 ml of phenol:chloroform:isoamylalcohol.
6) Add acid-washed or zirconium beads, so the liquid is 2-3 mm above the beads. Break the cells open by vortexing.
7) Add 0.2 ml of TE (pH 8.0). Vortex 15 seconds, making sure that everything is mixed.
8) Spin down at13,000 rpm for 5 min. Transfer the top aqueous layer to new tube. (The previous tube should be disposed of in a phenol bin.)
9) To the aqueous fraction in the new tube, add 1 ml 100% ethanol and mix by inversion. Leave at -20°C for 30 minutes.
10) Spin down at 13,000 rpm for 10 min, discard supernatant. Resuspend the pellet, which is mostly RNA at this point, in 0.4ml of TE; add 3µl of a 10mg/ml solution of RNAse A.
11) Incubate for 15 minutes at 37°C. Add 10µl of 3M sodium acetate pH 5.2, mix, add 1 ml of 100 ethanol, and mix again by inversion.
12) Spin down at 13,000 rpm for 10 min, discard supernatant. Air dry the pellet, wash with 70% ethanol, spin down again and remove the ethanol. Air dry again and resuspend pellet in 50 µl of water. The final DNA concentration should be around 0.5 µg/µl.
News alert: This protocol is spreading in other yeast groups that also work in our lab…the DIY approach is catching on, swifter, higher, and stronger!
In a recent article, I gave some tips about how to obtain good results with sequencing DNA after bisulfite conversion (it contains some tips that apply to the approach described in this article, too). Bisulfite sequencing is a very useful technique if you want to know the methylation status of every CpG in your genomic […]
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