We did the PCRs directly from cultured cells, without lysis. Our cells were adherent, so we washed them in PBS, scraped them and added to PCR mix. In the PCR program, we added ``hot start`` steps: 3 cycles of 95 degrees C for 3 min and 55 degrees C for 2 min, followed by normal PCR program. We used QIAgen hot start Taq polymerase. By doing this, you don`t need to use proteinase, because cells burst during the hot start step,  all the proteins are inactivated and DNA is released in the mixture. Of course, you will need hot start polymerase.

Thats a tricky one becuase streamlining as you have suggested might lead to crude lysates that will not PCR amplify efficently especially from a small starting cell mass

In the past I routinely extracted genomic DNA by proteinase K and SDS followed by column purification for the purposes of PCR genotyping  but admittedly that was for small sample numbers. you are attempting to stream line the protocol because of large sample numbers. Short of using vacuum manifolds in conjunction with genomic DNA purification kits Im not sure how you would proceed

Thanks. The title of my question was apparently modified beyond recognition, so let me explain a bit more :) The protocol I presented works perfectly fine, but I would like to optimize it further to get rid of the tube-opening and pipetting steps to add protK in between.

Not quite sure how to help. Good luck

Not sure if this would help, but maybe you could use an adhesive foil to seal your 96 well plate during your procedure?

You can use NaOH for genomic PCR without the need to treat your cells with SDS and Proteinase K. I used it longtime ago on S.pombe but the method works even with mamalian tissues (see article). Almost no handling! If you very little cells than maybe you can first perform WGA with phi 29 and then PCR

Are they adherent cells? If so, you can aspirate the media and wash the cells a couple of times with a small volume (100µl) of PBS. This will remove all media. Then I would add a buffer like 10 mM EGTA (maybe 50-100µl) and scrape the cells off the plate with pipette tip. Place the cell suspension  into a microfuge tube  and heat to 95°C for 10-15 minutes, centrifuge and use. Ensure the volume of template used is at least 1 tenth total volume of PCR reaction. Have used this before and works OK for genomic.

I have used NaOH method in the past. You must find the optimal volume for your average number of cells or it won't work (your DNA will be too dilute). For routine lysis of small tissue I use 100-120ul 50mM NaOH, 95C for 20-30mins, then neutralize with 1M tris pH~7.5, 20-30ul. Super easy, but you will have to play around volumes like I said. Maybe try more like 40-50ul NaOH.

A while ago I had to extract DNA from HEK293T cells for PCR, but was able to make a master mix at the start of both the lysis buffer (which I believe was 0.5% tween 20 in TE buffer) and proteinase K, of which was added to all the cell samples at the begin of the lysis (the cells had been spun down at 2000 rpm and all media removed, do there was no chance of interference). The lysis solution containing cells was then incubated at 1 hour for 55oC, followed by incubation at 95 oC for 10 minutes to denature the Proteinase K.

We were starting with slightly larger cell amounts, but this method only involved opening the tubes at the start of the protocol, and has been successful in our lab both for extract DNA from human cell lines and mouse ear punches for different PCR. While we did have to add the detergent Tween, given it is a relatively low amount it did not seem to interfere with the sebsequent PCRs.

Use Phire Animal Tissue Direct PCR Kit from Thermo Scientific. It may reduce your time and labor. It can perform well at presence of high inhibitor levels.

All the best.

Remove media.  Add 25ul "Quanta" extraction reagent.  Heat 75C 15 minutes.  Add 250ul water.  PCR 5ul in 20ul reaction.

now new kit from Thermo Fisher is available for direct PCR from sample without purification etc. pl look for it. might be interesting.

I heard this kit is pretty good for a low number of input cells for DNA extraction. There is usually a low number of pipetting steps with most kits too. I know there is a slight trade off with a slight loss in yield, but samples can always be concentrated and then pooled (see below).

https://www.promega.ca/products/dna-and-rna-purification/genomic-dna-purification-kits/maxwell-16-system-dna-purification-kits/maxwell-16-cell-lev-dna-purification-kit/

Hope this helps!

We did the PCRs directly from cultured cells, without lysis. Our cells were adherent, so we washed them in PBS, scraped them and added to PCR mix. In the PCR program, we added ``hot start`` steps: 3 cycles of 95 degrees C for 3 min and 55 degrees C for 2 min, followed by normal PCR program. We used QIAgen hot start Taq polymerase. By doing this, you don`t need to use proteinase, because cells burst during the hot start step,  all the proteins are inactivated and DNA is released in the mixture. Of course, you will need hot start polymerase.

There are a few heat-stable proteases out there too, if that turns out to be necessary. No experience with them though, sorry.

Blood cells are perfectly lysed by a couple of freezing-thawing cycles (even if just one cycle proves to be enough in most cases). This can also bee applied to other cells not associated to inter-cellular hard matrix (for instance scrapped from culture cell flasks). A simple chelex DNA extraction protocol can then be used prior to PCR. You only need to add the chelex resine suspension: just one pippeting.

Maybe you could improve DNA accessibility by adding histone deacetylase inhibitors in your cell media. Maybe you could do PCR without Proteinase K.

This works really good in my hands:

DirectPCR Lysis Reagent (cell) 100ml

Cat. No 302-C

It is from Viagen, easy to google.

I add 0.2 mg/ml protK (from a 1 mg/ml stock) to the lysis buffer right before use, and incubate o/n at 56C and thereafter 45 min 85C. It can't get easier to my knowledge.

Why do you have a 10 min 95C step before adding proteinase K? Seems unnecessary as ProtK should do the job added directly to the cells, particulariy if you add a tiny amount of detergent and perhaps prolong the 56C step. The initial lysis step may actually degrade DNA.

Mikael, I would recommend a Direct PCR kit --- I've used the Terra Direct PCR Polymerase kit and it works great, even if you only have very minimal template DNA to extract. If you do not need the combined extraction/PCR kit, there are many that you can get for fairly inexpensive prices that have the lysis buffer and proteinase K in the kit.

Chris

Can virus protein caspids  be perfectly lysed by DirectPCR Lysis Reagent  without protein k?