DNA extraction from sputum

[Zainul Zain]

Hi, netters;

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 
advice......v.v.....)

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 
prior use.



Thanks in advance.


Tang
Medical School
University Sains Malaysia
Kelantan, Malaysia