Mar 22, 2022
Genomic DNA prep with Quick Extract (QE) buffer
This protocol is a draft, published without a DOI.
- 1University of Edinburgh
Protocol Citation: Hannah.Long 2022. Genomic DNA prep with Quick Extract (QE) buffer . protocols.io https://protocols.io/view/genomic-dna-prep-with-quick-extract-qe-buffer-b6kkrcuw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 22, 2022
Last Modified: March 22, 2022
Protocol Integer ID: 59756
Keywords: genomic DNA, gDNA, QuickExtract, QE, clones, screening
Abstract
This protocol describes a high throughput extraction method to isolate genomic DNA for screening clones (e.g. from 24 well plate).
There are two options: i) you can split clones onto two plates and extract gDNA from one of those replica plates, or ii) during the process of splitting cells, you can keep some material for genotyping. This protocol describes the second option.
This protocol is for hESCs, but could work for other cell-types too.
Materials
QuickExtract (QE) buffer
Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane MatrixCorningCatalog #356231
ReLeSR™ 100 mL
Stemcell TechnologiesCatalog #5872
mTeSR™1 500 mL Kit
Stemcell TechnologiesCatalog #85850
QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050.
Before start
Grow cells to around 50% confluency.
Grow clones on a multi-well format, e.g. 24-well plate.
Prepare new matrigel coated plates for passaging clones and label PCR strip tubes corresponding to the position of the clones on the plates. E.g. 1-A1 would refer to plate 1, position A1 on the plate.
When clones are ready to be split, remove media and add ReLeSR for 00:00:30 . Aspirate ReLeSR and leave in incubator for 00:03:00 (up to 5 minutes) until colonies appear cracked.
3m 30s
In the meantime, prepare new plate by removing matrigel and adding some warmed mTeSR1 media.
Add appropriate amount of warmed mTeSR1 media for splitting, and tap plate gently. Using a P1000, gently scratch well to release remaining cells and put half media/cells in PCR tube and half in new plate.
Tap new plate to distribute cells and return to incubator. Monitor the following day and replace media (may see a lot of death).
Pulse spin strip tubes on bench-top mini-centrifuge. You may see pellet of cells. Remove media and replace with 50 µL QuickExtract buffer. Tap to dislodge pellet. Could pipette up and down.
Place in thermocycler with the following settings:
65°C 8-10 min
98°C 10 min
DNA samples are now ready to be used for PCR genotyping. For example, first try 1uL sample in a 10uL reaction (e.g. GXL PrimeStar - or Q5 polymerase). If PCR fails, try a dilution 1:5 of DNA, then 1:25 dilution etc.