Category: Genotyping

Hey HotSHOT

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).

Thoughts on genotyping.

First week back after the Christmas shutdown, and all my mouse strains have had litters weaned. The couple of hours it took to earpunch the 70-odd animals was inevitable, if tedious, and can be chalked up as a necessary requirement for working with transgenic mice. However, after extracting DNA from all those samples and then at least one PCR for each, I started to wonder how necessary the endless hours of pipetting might be.

Our University has an agreement with an external paid genotyping service, which some researchers in the animal facility have been using. However, our lab as a rule haven’t used it, because it is expensive, and we have always thought it would cost too much to get all our mice genotyped this way. Also, I’ve always maintained that genotyping builds character, which is why I often allow new students and technicians the opportunity to learn using my samples.

Anyway, given the time investment needed to successfully genotype the mice, I wanted to compare the actual cost per sample of using the automated service compared to doing it ourselves, including the cost of reagents and postdoc salary time.

Looking back through my genotyping lab book, my usual batch of DNA extractions involves around 35.85 samples, and my PCR’s average 26.35 samples. In the table below, I have estimated the various costs associated with genotyping this typical batch, including the likely time spent by yours truly, that could otherwise be spent on more productive lab activities. I have estimated my time to be worth 22.9 £/hour, based on my salary and working 37.5 hours per week for 47 weeks a year (which is my contracted time, minus some of my annual leave that I never end up taking anyway).

Obviously, this calculation is heavily dependent upon the number of samples run at a time, so delaying the genotyping to do bigger batches would save on time/costs (see graph below for estimated drop in cost per sample with increased run sizes). But, it is possible that we would end up paying more for the mouse costs, so that needs to be kept in mind.

Now, comparing to the genotyping service, they charge $7.85 per sample (at current exchange approx. £5.77). This costs about double my calculation for doing it myself, and correlating on the cost/sample graph shows that this lines up slightly below 15 samples, so unless the genotyping batches decrease in size, I think it is unlikely I will be able to persuade the boss to fork over the extra cash to pay for external genotyping services.

However, there is another factor to take into account, which is the current *situation* the world finds itself in, namely Covid19. We (in the UK) are currently in our third national lockdown due to rampant infections, so it might make sense from a health and safety perspective to spend a bit extra on contracting the genotyping externally, if it would save workers from having to come in to the lab and mix in a possibly Covid-transmissable manner. Just something to think about.