DNA Isolation from Tails (and ES Cells)
1. Prepare lysis buffer by diluting Proteinase K stock to 250 ug/ml (cells) or 500 ug/um (tails) in appropriate quantity of buffer (500ul per sample)
2. Add 500-ul buffer to each tail sample or well of cells (this is for a 6-well plate, adjust to smaller or larger volume for other plate sizes).
3.Incubate tail samples overnight at 37ƒ C or cell samples 2 hours to overnight at 37 C. Transfer cell samples to eppendorf tubes after incubation.
4.Add 250 ul saturated (6M) NaCl to each tube.
5.Vortex 2-5 min.
6.Place on ice for 10 min.
7.Spin 10 min. at low speed (9500 rpms on Eppendorf microcentrifuge)
8.Carefully remove top 500 ul to fresh tube with 1 ml of 100% EtOH and mix by inversion.
9.Centrifuge at high speed for 10 min. (14,000 rpm on Eppendorf microcentrifuge)
10.Remove supernatant by vacuum and wash with 70% EtOH 2X to remove salts (200 ul/wash). Dry.
11.Resuspend in 50-100 ul of TE or H2O O/N at room temp.
|Reagent||per 50 mls:||Final Concentration|
|1 M Tris, pH 8||500 ul||10 mM|
|5 M NaCl||1 ml||100 mM|
|0.5 M EDTA, pH 8||1 ml||10 mM|
|10% SDS||2.5 ml||0.5 %|
|H2O||to 50 mls|
Proteinase K: Final Concentration for: 1. ES clones 250 ug/ml
2. Tails 500 ug/ml
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