Below is the brief details of what i done.
Step1: Spin down 10ml of yeast cells suspension
Step2: Resuspend the cell pellet in 1ml of autoclave H2O.
Step3: Place the cell suspension in centrifuge tube containing glass beads.
Step 4: homogenized using biospec minibead beater for 40seconds and incubate in ice for 1min
Step5: repeat step 4 for 5 times
Step6: spin down the cell debris and glass beads
Step7: Pipette the suspernatent into a new centrifuge tube and add equal volumn of USB corporation phenol chloroform isoamylalcohol( 25:24 :1)
Step8: invert the tube for a few times
Step9: spin at 14000xg for 10mins
Step 10: carefully pipette the aqueous layer and transferred to a new centrifuge tube
Step11: add 1/10 vol of sodium acetate and 3vol of absolute ethanol to the aqueous layer
Step12: put in ice for 30mins
Step 13: spin down the precipitation and decant the supernatent
Step 14: air-dry for 5mins
Step 15: add 1ml of autoclave water to the precipitate
Discussion: i follow abv protocol and found that i get 2 distinct band (RNA band) and very little DNA on the wells of the gel after running a gel. i heard that the phenol-chlorform should be pH8 to tune to dna extraction, so i use a pH indicator strips to test the commercial PCI i used and found it was around 8. And as for the sodium acetate, it is 3M and pH 5.2( using HCl to calibrate the pH).
In addition, i suspect that the DNA might be in the chloroform layer, hence i also add 1/10 vol of sodium acetate and 3 vol of abs ethanol to the chloroform layer and try to precipitate it and after nanodrop spec measurement, it was 3k++ ng/ul and 260/280:1.96 , however nothing appear in the gel.
And now i seriously what is gone wrong
sorry for my bad english.
Edited by Johnry, 10 November 2009 - 02:44 AM.