I was doing a massive round of genotyping yesterday, and was happy to have a rapid DNA extraction method. It reminded of the summer of 2014, when my supervisor had been awarded 3 grants. So we had 3 postdocs due to start at the same time, at the beginning of October. In preparation of the new arrivals, we had acquired a few new transgenic lines and were breeding up a bunch of crosses to produce experimental animals.
This was great news all round, except that come July two PhD students and a postdoc had recently left, and the only other person in the lab was our technician. And we now had hundreds of mice being weaned that needed genotyping. I ended up ear punching a dozen or more cages of mice a day for weeks on end, and with tremendous help from our technician, we were running hundreds of PCR reactions a week.
Ok, so where am I going with all this? The part of this process that took the longest was extracting DNA from the ear notches to use for PCR, and I was getting annoyed by the time-intensive nature of it.
We used a proteinase K digestion overnight, followed by isopropanol precipitation and cleaning with ethanol before reconstituting in water, and it took hours. And we were doing this every day, for weeks. So, I started looking around for quicker and easier methods – I was quite sure we didn’t need such high quality sample prep for a genotyping PCR.
I found a method published in 2000 (and it turns out everyone uses this because it’s so quick and easy, but I had never heard of it), which used alkaline and high heat to degrade the tissue, followed by an acidic buffer mixed in to neutralise the alkali1.
Just in case any readers haven’t come across this method before, I cannot recommend it highly enough. We use it routinely now, and there’s only a couple of PCR’s we run that require a cleaner sample, and then we do our old long extraction.
A quick summary of the rapid DNA extraction protocol:
- Lysis buffer (for 50 ml)
- 20 µl of 0.5 M EDTA
- 125 µl of 10M NaOH
- 50 ml MilliQ water
- Neutralising buffer (for 50 ml)
- 315 mg of Tris HCl (don’t use pre-buffered Tris solution)
- 50 ml MilliQ water
- Place tissue sample (eg. ear notch) in a 1.5 ml eppendorff
- Spin the tissue samples at 13,000 rpm for 30 s to get tissue to bottom of tube
- Pipette 75 µl of lysis buffer onto sample and place on 95 C heat block for 45 mins
- Place samples on ice for 5 mins, then briefly spin contents to bottom of tube
- Pipette 75 µl of neutralising buffer onto sample and vortex
The whole protocol only takes about an hour, and then the samples are ready to use for PCR. Unfortunately, I only came across this method after many many (MANY) hours spent doing our old slow extraction method. It does, however, continue to save us a lot of time in the lab, so I’m happy I found it, and maybe it will help someone else out there save time as well.
1. Truett et al. Biotechniques 29(1), 52-54 (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT).