help with the Quickextract solution from Epicentre

[Elphège Nora]

Hi all, 

A few protocols have recommended the use of the Quickextract solution from Epicentre to prepare genomic DNA.

I have used it to prepare DNA from cells grown in 96well plates with mitigated success. I am using 20µL of the solution per well, vortex the plate, heat at 65°C for 6 min, vortex the plate, heat at 95°C, cool down and then use 1µL for a 20µL PCR reaction.

Sometimes that works beautifully, sometimes not at all. Also primers that used to give me very clean bands on ethanol precipitated DNA start showing extra bands somehow. Also I find that the nanodrop reading hard to trust since the absorbance spectra tend to show extra peaks - probably because of remaining traces of culture medium.

How much solution do you guys typically use for a 96well and how much volume of that do you use for your PCR? Do you find that you need to dilute the DNA and do you usually have to adjust your annealing temperatures compared to classical extraction protocols? 

I would really appreciate any recommendation/protocol to streamline DNA extraction from 96w plates. 

Thanks a lot in advance!

Elphege

 

[Patrick David Hsu]

Hi elphege,

Here is the protocol we use:

65C 15:00

68C 15:00

98C 10:00

For 96-well plates, I use 20 uL of solution per well. You definitely will get some culture media left over but it's fine. I just dump media out of the wells, either with a multichannel vacuum or simple inversion. Dry on kimwipes by tapping and multichannel in the QE. Pipet-mix before it becomes viscous and transfer to a PCR plate. Run the program above.

The nanodrop reading obviously doesn't reflect the DNA concentration because it's not pure, but with an H2O blank, I add ~500 ng of QE (by nanodrop readout) per 50 uL PCR reaction. This is based on my empirical estimates from titrating different input template amounts.

My PCR primers are designed using IDT oligo analyzer or Northwestern's Oligo Calc (for calculating Tm). I aim for Tm 60C with the program, and I use the same Tm with Herculase II polymerase from Agilent. I also use KAPA Hifi from KAPA biosystems, but I use Tm 63C for those PCRs.

Hope this helps

Patrick

[Matthew Thompson]

The extraction method I have had success with includes dissociating cells with TrypLE, centrifuging for 5 minutes at 300xg, aspirating (NOT inverting after centrifugation), and then gently washing once with PBS to remove traces of medium. The cells stick pretty well through the PBS wash. Then, after adding 20 µL of QE, I perform the 65, 68, 98°C treatment directly in the culture plate on a heat block, and then spinning down to collect the condensation. This saves a rack of pipette tips and a PCR plate, but requires a little more hands-on time and attention. I dilute the resulting QE solution 1:10 in water and use 1 µL in a 25 µL PCR. This consistently yields good PCR products for me with NEB's OneTaq Quick-Load Hot Start master mix using a TD-PCR protocol and avoids the need to spend time finding the concentration of each well on a NanoDrop. Subsequent direct RFLP in 50 µL reactions for identifying positive HDR works as well without having to purify the PCR product.

Once, I forgot about the plate and let it sit at 98°C for a little over an hour, but PCR and RFLP still worked using the 1:10 diluted DNA.

Ideally, I'd like the protocol to look like this: remove medium, wash once with PBS, add 20 µL QE, incubate at 65°C for 30 min (or less), 98°C for 10 min, and dilute 1:10. I have not tried this yet though since I wasn't sure if skipping the dissociation would cause problems with other steps. 

Buying a programmable heat block would help too...not so sure that will fly though.

[Phil Abbosh]

Elphege
One thing i tried was making my own lysis buffer that i add to a plate and that seemed to work pretty well.  i cloned my plate into another plate with media+10% DMSO to put in -80C while i screened wells:

wash plate with PBS, invert onto paper towels as Patrick said (assuming you have adherent cells), then I just used 1% triton X100 as the lysis buffer (50 uL per well).  instead of heating, i just freeze/thaw 4 times in a -20C.  if there is still some debris it didnt seem to inhibit my PCR reaction. 1 uL of lysate in a 25 uL PCR reaction gave pretty reliable results for a screen that i could go back and thoroughly work up later.  I use accuprime PFX, life technologies. 

i tried several different triton concentrations, and NP40 and SDS at different concentrations too.  Triton worked the best but you might try some others just to see what works for you (I just used 1000 cells in an eppy tube instead of a whole plate to optimize the protocol).  i didnt mess with buffering, EDTA, proteinase K etc but that might have made it better.  the ideal protocol is like matthew's: no changing plates and little manipulation to get DNA that is adequate for your workup.

phil

[Ralitsa Radostinova Madsen]

Hi Matthew,

Washing once with PBS and adding the QE straight to the wells, pipetting up-and-down and adding some nuclease-free H2O to make the sample less viscous before collecting into a tube, is what I do, and it has worked pretty well so far. So skipping the dissociation shouldn't cause you a problem; essentially, you just want the DNA out - it is a crude prep anyway. 

BW, 

Ralitsa