Publisher

National Public Health Institute

DNA:n eristys ja säilytys

Fenoli-kloroformi eristysmetodi/Method for DNA-extraction

Method for DNA-extraction / Department of Molecular Medicine /National Public Health Institute / Helsinki, Finland / M. Perola/1997 

Blood should be drawn in an EDTA-tube (the one with a violet cap). NOTES: -Avoid vortexing the sample at any point (damages DNA) -remember sterility, use only sterilized equipment at any point Bloodtube must be freezed to -20 C for couple of days before the following. DO NOT USE FRESH BLOOD (e.g. not frozen). DO NOT
FREEZE AGAIN -if necessary, the method can be stopped at point 3 (pellet) and frozen for several days. 
1. Add 40 ml of Buffer A1 to 10 ml of EDTA-blood Mix well, keep in ice for 10 minutes, mix once in a while. Centrifuge 30 minutes 1500 rpm at +4_ C Leukocytes form a small pellet at the bottom of the tube. Pour supernatant away as well as possible, carefully so that the pellet won't fall off. 
2. Repeat point 1.
3. Add 2 ml of Buffer B2 on leukocyte pellet and try to break the pellet by pipeting it back and forth. 
4. Add: 500µl 1mg/ml proteinase K and 100µl 20% SDS 
5. Incubate at +37_C overnight (12-16 h) 
6. Pour samples in geltubes3. 
7. Add 2ml of buffered phenol (pH about 8) and 2 ml of chloroform/isoamylalcohol mixture4. Mix well, do not vortex 
8. Centrifuge at less than 20_c 5 minutes at 2000 rpm. The gel goes in between the sample and phenol/choloform. 
9. Pour supernatant (containing the DNA) into sterile, small glass vials. 10. Add 200ul 2M KCL and mix gently. 
11. Pour 5ml of ethanol carefully on the sample-KCL mix so that the phases preferably do not mix 
12. Shake gently so that the DNA visuliazises at the border of the phases. Roll it on the glass stick by rotating the stick. 
13. Air dry the stick for 10-20 minutes 
14. Put the glass stick, where the DNA has dried on, in 500µl of TE-buffer for 20 minutes. Make sure that all DNA gets off the stick before disposing of it. 
15. Let soak for a day, then measure ABS260 and ABS280 (1:80). 
16. Concentration="ABS260" x 80 x 50="µg/ml" Purity="ABS260/ABS280" 

FOOTNOTES: 1. Buffer A: 320 mM Sucrose 1% Triton X-100 5 mM MgCl2 10 mM Tris-HCl pH 7.6 2. Buffer B: 25 mM EDTA pH 8.0 75 mM NaCl 3. Geltubes: Phase Lock Gel" IIB Light 5 Prime--> 3 Prime, INC. ®, CO, USA 4. Choloform/isoamylalcohol mixture: 4% isoamylalcohol 96% choloform 

References: Vandenplas S, Wiid I, Grobler-Rabie A, Brebner K, Ricketts M, Wallis G, Bester A, Boyd C, Mathew C (1984): Blot hybridisation analysis of genomic DNA. J Med Genet 21: 164-172.