Need protocol for extracting DNA from amniotic fluid - (May/27/2006 )
does anyone have a good protocol for DNA extraction from human amniotic fluid ?
This protocol works well (standard phenol:chloroform extraction). I assume you're talking about whole amniotic fluid, not cultured cells??
You can leave out step 6 and step 11 if you like. The same protocol is used for cultured cells but increase the amount of lysis buffer and other reagents accordingly in line with increased pellet size. Good luck.
Extracting DNA from whole amniotic fluid (amniocytes).
1. Divide the sample of amniotic fluid into labelled 2 mL eppendorf tubes (the number of tubes will depend on the volume of sample received). Spin in microcentrifuge at 13,000 rpm for 10 min. Alternatively, centrifuge the cells in a larger tube, remove supernatant and transfer entire pellet to single eppendorf using PBS and then centrifuge eppendorf as above.
2. Remove supernatant.
3. Add 375 uL salting out buffer, 6.25 uL 20% SDS and 125 uL proteinase K (5 mg/mL)
4. Incubate O/N in 37C water bath or 30 - 60 min at 65C (flicking to resuspend constantly) to digest protein.
5. Label 3 X 1.5 mL microfuge tubes
6. To the digested sample add 400 uL phenol, invert and spin at 10,000 rpm in microcentrifuge for 5 min.
7. Transfer aqueous layer to another tube and add 400 uL 1:1 phenol:chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
8. Transfer aqueous layer to a new tube and add 400 uL chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
9. Transfer aqueous layer to a new microfuge tube and add 1/10 volume 3 M NaAc, pH 5.2 (40 uL) and 2.5 volumes of 100% ethanol (1 mL). Invert tube gently to precipitate DNA.
10. Spin at 10,000 rpm for 5 min and pour off ethanol.
11. Rinse the pellet once with 70% ethanol, centrifuge 10,000 rpm for 5 min. (Do not include this step if the pellet is very small)
12. Dry DNA pellet in laminar flow cabinet and resuspend in 20 - 50 uL of sterile water depending on size of pellet.
Salting out Lysis Buffer
1M Tris pH.7.5 10 mL
5M NaCl 80 mL
0.5 M Na2 EDTA 4 mL
Add dH2O to 1lt. and mix reagents with magnetic stirrer for short while. Label and date. Autoclave. Store at RT.
thanks alot karyotyper ,,, i well try it