DNA extraction – miniprep from spores for PCR

Detail:

Miniprep from spores for PCR.

1. With a loop, pick conidia from a single colony and resuspend in 0.8ml of 
sterile water until suspension becomes turbid. 
2. Vortex and centrifuge to sediment conidia, discard supernatant. 
3. Add 0.1ml of buffer1. 
4. Add 0.1ml of buffer2. Mix well and incubate for 20 mins at 65o
C. Allow to cool 
at RT. 
5. Add 0.1 ml of buffer 3 and mix. Top speed centrifuge at 6o
C for 8 min. 
Without disturbing the pellet transfer 0.25ml of clear supernatant to a new 
tube and discard the rest. 
6. Add 0.15 ml of distilled water. Precipitate DNA with 40ul of isopropanol. 
Centrifuge at top speed for 20 min at 6o
C. 
7. Wash with 0.3ml of 70% ethanol. Centrifuge 5min at RT, discard 
supernatant. 
8. Allow the pellet to air dry for some minutes and resuspend pellet (invisible ) 
in 10 ul of distilled water. 
9. Use 2ul per PCR reaction in a final volume of 50ul. 
Buffer1 (resuspension) 
50mm Tris-HCl pH 8.0 
10mm EDTA 
100ug/ml Rnase A 
(store at 40
C.) 
Buffer2 (lysis) 
200mm NaOH 
1%SDS 
Store RT 
Buffer3 (neutralisation) 
3.0M Potassium acetate pH 5.5 
store RT 
Method of Dr J A Calera-Abad, kindly supplied by Raquel 
Lopez:Raquel@imb.usal.es 

Laboratory Protocols