DNA Isolation from ES Cells -96 well plate


Posted by cpark
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Protocol

Bradley Lysis Buffer without Proteinase K
Stock Solutions                      Final Conc               for 500mL
1M Tris-HCl (pH 7.5)               10 mM                        5ml
0.5M EDTA                            10 mM                        10 mL
10% SDS                              0.5 %                        25 mL
5M NaCl                                10 mM                       1ml
H2O                                                                 bring up to 500ml
 
ProteinaseK (20mg/mL)           
:add just before use as 1mg/ml final concentration
                                   
EtOH/NaCl Mix
100% EtOH                  394 mL
5M NaCl                        6 ml
Total volume                400mL

Methods: 
1. Grow ES cells in a 96-well plate to be over-confluent.
2. Quickly invert the plate over to dump media and remove excess liquid by blotting
on paper towels.
3. Wash cells 1x with 150 uL PBS, then dump PBS.
4. Add 50 ul of Bradley Lysis Buffer containing proteinase K.
5. Replace lid and seal the plate with parafilm.  Put the plate into a humidified
chamber
6. Incubate in the humidified chamber O/N @ 60 degree.
7. Allow the plate to cool to RT

8. Add 100 uL ice-cold EtOH/NaCl mix to precipitate DNA and mix well. Then
incubate the plate about 30 minutes at RT
9. Spin in 96-well plate holder centrifuge, 3000rpm 20 minutes
10. Invert the plate to decant liquid.   Blot the plate on paper towels to remove access
liquid.
11. Add 150uL cold 70% EtOH and spin 10minutes at 3000 rpm to rinse the pellet.
Decant supernatant onto paper towels.
12. Repeat washing step ( #11) and air-dry DNA pellet until there is no detectable
EtOH smell (approx 10-15 minutes)
13. Add 30 uL of warm TE pH 8.0
14. Cover Plate with wax cover and incubate at 56 degree ~10 mins

 

Materials: 

 

Bradley Lysis Buffer without Proteinase K

Stock Solutions                      Final Conc               for 500mL

1M Tris-HCl (pH 7.5)               10 mM                        5ml

0.5M EDTA                            10 mM                        10 mL

10% SDS                              0.5 %                        25 mL

5M NaCl                                10 mM                       1ml

H2O                                                                 bring up to 500ml

 

ProteinaseK (20mg/mL)           

:add just before use as 1mg/ml final concentration

                                   

EtOH/NaCl Mix

100% EtOH                  394 mL

5M NaCl  6 ml

Total volume                400mL

Methods: 
  1. Grow ES cells in a 96-well plate to be over-confluent.
  2. Quickly invert the plate over to dump media and remove excess liquid by blotting on paper towels.
  3. Wash cells 1x with 150 uL PBS, then dump PBS.
  4. Add 50 ul of Bradley Lysis Buffer containing proteinase K.
  5. Replace lid and seal the plate with parafilm.  Put the plate into a humidified chamber
  6. Incubate in the humidified chamber O/N @ 60 degree.
  7. Allow the plate to cool to RT
  8. Add 100 uL ice-cold EtOH/NaCl mix to precipitate DNA and mix well. Then incubate the plate about 30 minutes at RT
  9. Spin in 96-well plate holder centrifuge, 3000rpm 20 minutes
  10. Invert the plate to decant liquid.   Blot the plate on paper towels to remove access liquid.
  11. Add 150uL cold 70% EtOH and spin 10minutes at 3000 rpm to rinse the pellet. Decant supernatant onto paper towels.
  12. Repeat washing step ( #11) and air-dry DNA pellet until there is no detectable EtOH smell (approx 10-15 minutes)
  13. Add 30 uL of warm TE pH 8.0
  14. Cover Plate with wax cover and incubate at 56 degree ~10 mins