TITLE: Preparing of genomic DNA from in vitro cultured cells
AUTHORS: Hanqin  Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
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
1. 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]
SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
1. Add 500 µl crude lysis buffer (Proteinase K added) to a fully confluent well of a 6 -well plate (~1.5 million cells) 
SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
2. Incubate at 37 °C 10 min 

SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
3. Collect the crude lysate into a 1.7 ml Eppendorf tube.
SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
4. Incubate at  50 °C   

SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
5. Transfer 50 µl to a 200 µl microcentrifuge tube
SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
6. Heat inactivate at 95 °C  for 5 min  in a thermocycler.

SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
7. Chill on ice 

SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
8. The crude cell lysate is ready for PCR
SECTION: A.	Preparing crude cell lysate directly from hPSCs culture
1.1.  
Crude lysis buffer
 


SECTION: B.	Preparing crude cell lysate from dissociated cells
9. Wash cells with DPBS once
SECTION: B.	Preparing crude cell lysate from dissociated cells
10. For each well in a 6-well plate, use 0.5 ml Trypsin
SECTION: B.	Preparing crude cell lysate from dissociated cells
11. Incubate 37 °C 5 min 

SECTION: B.	Preparing crude cell lysate from dissociated cells
12. Inactivate Trypsin using 2 ml wash medium 
SECTION: B.	Preparing crude cell lysate from dissociated cells
13. Centrifuge at 200-300 x g, 5 min  

SECTION: B.	Preparing crude cell lysate from dissociated cells
14. Remove supernatant
SECTION: B.	Preparing crude cell lysate from dissociated cells
15. 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.
SECTION: B.	Preparing crude cell lysate from dissociated cells
16. Transfer the crude lysate into a 1.7ml Eppendorf tube.
SECTION: B.	Preparing crude cell lysate from dissociated cells
17. Incubate at  50 °C   

SECTION: B.	Preparing crude cell lysate from dissociated cells
18. Transfer 50 µl to a 200 µl microcentrifuge tube
SECTION: B.	Preparing crude cell lysate from dissociated cells
19. Heat inactivate at  95 °C 5 min in a thermocycler

SECTION: B.	Preparing crude cell lysate from dissociated cells
20. Chill on ice 

SECTION: B.	Preparing crude cell lysate from dissociated cells
21. The crude cell lysate is ready for PCR
SECTION: B.	Preparing crude cell lysate from dissociated cells
12.1. Wash medium
 

SECTION: C.	Preparing cell lysate from FACS-sorted cells (small cell number)
22. 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.