2 replies to this topic

[Johnry]

i am new in molecular work. I have some problems regarding the extraction of DNA using P:C:IAA (25:24:1)

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.

[Prep!]

why dont u just add RNAse to remove the RNA contamination!!!

[phage434]

Are you trying to isolate plasmid DNA or genomic DNA? I suspect you may not be mixing the initial phenol/chloroform with the bead supernatant sufficiently. I would vigorously vortex at this stage unless I was concerned about shearing of genomic DNA, and then I would not be using a bead beater to isolate it. I would suggest that after the initial phenol chloroform extraction that you extract again with chloroform or chloroform/IAA only to remove phenol. Do you see a DNA pellet after precipitation? You should be washing the DNA with 70% ethanol, spinning it down, prior to drying it You may be failing to resuspend the DNA after precipitation. It does not necessarily redissolve easily, especially if overdried, or if it is genomic DNA. You should resuspend in TE rather than water, which will make resuspension easier, and allow at least a few hours at 50-55 C with shaking.