DNA extraction from sputum
zainul at KB.USM.MY
Sun Jun 26 03:24:57 EST 1994
Does anyone know any fast and reliable method for extracting DNA from
sputum for PCR???
The method I am using now is as follows:
(I am the problem of getting enough DNA for amplification. Please
1. Homogenised sputum with equal vol. of 0.5M NaOH, 0.05M
Sodium Citrate at RT for 15 mins.
2. Spin 3500 rpm/15 mins
3. Wash pellet with phosphate buffer pH 6.8
4. Resusp. pellet in 2 ml of 50mM Tris.HCL pH8.3 and aliquot 500ul each.
Keep at 4C prior use.
5. Take ONE 500ul aliquot and process as follows:
6. Heat inactivate at 80C/20 mins
7. Spin 12,000g/5 mins, flick off sup.
8.Emulsified pellet by vigorous flicking in 50ul Chloroform.
9. Add 50ul of H2O to emulsified with Chloroform
10. Spin 3 mins in a m/fuge tube. Use 5ul for 20ul PCR reaction. Store
the rest at -20C without removing Chloroform and respin briefly
Thanks in advance.
University Sains Malaysia
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