Preparing of genomic DNA from in vitro cultured cells
Yogendra Verma, Hanqin Li, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes a standard procedure for preparing crude cell lysate which can be further analyzed by PCR.
Protocol overview:
A. Preparing crude cell lysate directly from hPSCs culture
B. Preparing crude cell lysate from dissociated cells
C. Preparing cell lysate from FACS-sorted cells (small cell number)
General notes
- Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Steps
A. Preparing crude cell lysate directly from hPSCs culture
Add 500 µl crude lysis buffer (Proteinase K added) to a fully confluent well of a 6 -well plate (~1.5 million cells)
Crude lysis buffer
| A | B |
|---|---|
| KCl | 50 mM |
| MgCl2 | 2 mM |
| NP-40 | 0.45% |
| Tween-20 | 0.45% |
| Tris | 10 mM |
| Proteinase K (add before use) | 250 µg/ml |
Incubate at 37°C 0h 10m 0s
Collect the crude lysate into a 1.7 ml Eppendorf tube.
Incubate at 50°C
Transfer 50 µl to a 200 µl microcentrifuge tube
Heat inactivate at 95°C for 0h 5m 0s in a thermocycler.
Chill On ice
The crude cell lysate is ready for PCR
B. Preparing crude cell lysate from dissociated cells
Wash cells with DPBS once
For each well in a 6-well plate, use 0.5 ml Trypsin
Incubate 37°C 0h 5m 0s
Inactivate Trypsin using 2 ml wash medium
Wash medium
| A | B |
|---|---|
| DMEM/F12 | 470 ml |
| Newborn Calf Serum | 25 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
Final volume; 500 ml
Centrifuge at 200-300x g
Remove supernatant
For a fully confluent well of a 6-well plate, mix the cell pellet with 500 µl crude lysis buffer (Proteinase K added). Reduce the amount of crude lysis buffer if the culture is not fully confluent.
Transfer the crude lysate into a 1.7ml Eppendorf tube.
Incubate at 50°C
Transfer 50 µl to a 200 µl microcentrifuge tube
Heat inactivate at 95°C 0h 5m 0s in a thermocycler
Chill On ice
The crude cell lysate is ready for PCR
C. Preparing cell lysate from FACS-sorted cells (small cell number)
For small cell numbers, we usually use a direct cell to DNA extraction kit (Exact N Amp Blood PCR Kit [Sigma, # XNAT2-1KT]) according to the manufacturers’ instructions.
