Sebastian Cocioba
posted 2 months ago
Amateur šŸŒæ Biologist ā€¢ Aspiring šŸŒ» Designer Independent Researcher at Binomica Labs Degreeless Heathen #diybio ā€¢ #homelab šŸ§¬šŸ“–: ATinyGreenCell.com šŸ¦£:@ATinyGreenCell@genomic.social šŸ¦:@ATinyGreenCell
Method

Ultra-cheap PCR Precipitation using PEG and Salt

I just stumbled upon an extremely cheap and simple way to clean up PCR products without the need for columns or fancy reagents. Here's my slight tweak of this wonderful Open Wetware entry.

PCR Precipitation Buffer

  1. To a 15mL tube, add 3g PEG 3350. (I use Miralax brand which works well)
  2. Add 2.19g NaCl.
  3. Fill to 12mL with distilled H2O and vortex thoroughly to dissolve.
  4. Top off to the 15mL mark. Vortex thoroughly to mix. Store at 4C; good for 30 days.

PCR Precipitation

  1. To a completed PCR reaction in a 0.2mL PCR tube, add 1:1 PCR Precipitation Buffer.
  2. Vortex briefly to mix.
  3. Incubate at 37C in a PCR machine for 15 minutes.
  4. Transfer to 1.5mL tubes and spin down 15mins at maximum speed.
  5. Discard the supernatant.
  6. Add 200uL of ice cold 80% Ethanol, vortex to mix, and spin down 10mins at max speed.
  7. Decant supernatant and spin down 1min at max speed to collect residual alcohol.
  8. Using a 200uL pipette, carefully remove residual solvent.
  9. Dry at 37C for 5mins.
  10. Resuspend in 20uL of DH2O, vortex thoroughly and spin down briefly to fully resuspend pellet.
  11. Quantify DNA concentration via Qubit, Nanodrop, and/or Gel Electrophoresis.

The gel image attached is from an actual precipitation I ran on two samples of PCR product. The gel order is:

Ladder PCR Reaction 1 Precipitation 1 PCR Reaction 2 Precipitation 2 Ladder

The amplicon was 2652bp and 10uL of either raw PCR reaction or purified sample was run in each lane. Yield looks very promising and cleanliness of the resulting amplicon is more than adequate for downstream work like sequencing and/or cloning. Enjoy!

15
Shege Reasons2 months ago

What is the max spin speed? 15000 x g?

Sebastian Cocioba2 months ago

@Shege Reasons, I use an eppendor miniSpin Plus which max speed is 14,500rpm which equates to 14,100g so at least 14,000 x g should work. As fast as your fuge allows, ideally.

Thanks for simplifying it / posting. I remember coming across this kind of thing in a kit, a tube marked PEG precipiation mix, and checking it out. It's a nice trick know.

There's also a version in Maniatis, just checked https://archive.org/details/molecularcloning0000samb/page/n81/mode/2up , I think they use slightly higher salt and lower and diff PEG, but not sure where the threshold is for precip

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