acid phenol:chloroform extraction - Im confused (Oct/04/2005 )

Hello,

I have to perform acid phenol:chloroform extraction of RNA post DNase treatment. I have found some protocols but the more I find the more confused I am
In some protocols you should use a mixture of phenol:chloroform:2M Na acetate(pH4):isoamyl alcohol is the proportions 250:49:25:1
I others a mixture of phenol:chloroform:isoamyl alcohol as 125:24:1

What is the difference? i thought maybe they are equivalent if in the second one acid phenol is used, so the Na acetate in the first is to acidify the mixture when you use neutral phenol (did this have any sense?
Also, what kind of phenol should I use?
And, what is the total volume of solution to use?

Hopefully someone who uses this method could give me some light

Thanks a lot,

coco

-coco-

The phenol should be equilibrated/saturated using 50mM sodium acetate pH4. The procedure for this is in Maniatis

I use Phenol:Chloroform:IAA 25:24:1,
followed by chloroform:IAA 24:1,
then add 0.1 volumes of 3M NaOAc pH5 & 2.5 volumes 100% Ethanol to precipitate

-John Buckels-

QUOTE (John Buckels @ Oct 5 2005, 11:13 AM)

i use the same protocol.
The one you mentionned above are for saving some times. Bt if you save time, you'll get bad quality of RNA and the extracted RNA will be degraded more than if you perform a longer but more cleaner protocol.

-fred_33-

QUOTE (John Buckels @ Oct 5 2005, 07:13 AM)

I've never followed with the 24:1 CHCl3:IAA. Have you ever omitted that step and noticed any major difference?

Thanks,
Hank

-haringsh-

I would still have something to ask

What amounts of these mixtures do you use for a certain amount of RNA?

Thanks again

-coco-

hi
when i omit this step, the aqueous phase still smells phenol.
i follow by 2 rinses of etoh 100% and two of etoh 70%
The protocol using chloroform iaa is cleaner for sure. But i've prepared plasmid ommiting the chlo IAA step and i get very clean sequencing by these preps. So i would say that a good wash do the job. But if i want a sure clean, i do chlo iaa
hope i'm not too confusing...
fred

-fred_33-

what Fred said

-John Buckels-

QUOTE (coco @ Oct 5 2005, 09:49 AM)

Hello,

I have to perform acid phenol:chloroform extraction of RNA post DNase treatment. I have found some protocols but the more I find the more confused I am :blink:
In some protocols you should use a mixture of phenol:chloroform:2M Na acetate(pH4):isoamyl alcohol is the proportions 250:49:25:1
I others a mixture of phenol:chloroform:isoamyl alcohol as 125:24:1

What is the difference? i thought maybe they are equivalent if in the second one acid phenol is used, so the Na acetate in the first is to acidify the mixture when you use neutral phenol (did this have any sense? :huh:
Also, what kind of phenol should I use?
And, what is the total volume of solution to use?

Hopefully someone who uses this method could give me some light :unsure:

Thanks a lot,

coco

Hello!
Maybe You should better use the premix "TRIZOL Reagent" from Invitrogen: http://www.invitrogen.com/content.cfm?pageid=469

-nwenta-