Hello all I am new to this procedure but I need to get a good protocol to preform acid phenol:chloroform extraction of RNA post DNase treatment. Are their any good references?
Thanks

Use QIAgene kit for RNA extractions. It has DNAse digestion option.

Well the RNA has been isolated using Trizol allready so I dont know if that kit will work, but I will check it out. In the meantime does anyone have a protocol that might work in this case? I would appriciate it.

RNeasy Kit (QIAgen) can be used to clean up RNA previously isolated by different methods or after DNA digestion.

1) Add an equal volume of Phenol:choloform:isoamyl alcohol solution to your RNA (I recommend keeping volume of RNA below 500ul or less in a 2ml eppendorf...add 500ul of phenol solution).

2) Vortex, spin till the two phases have separated (you might get a white intermediate phase)

3) aspirate the top layer (your RNA) and transfer to clean tube

4) add 1/10 volume of Sodium Acetate (3M, pH=5.2)

5) add 2x volume of 100% ethanol (this is why i limit volume/tube in step 1)

6) place at -80C for 1h (longer if your [RNA] is low)

7) spin at full speed at 4C for 20 mins.

8) dry pellet and resuspend in desired buffer.

Hello,

I have been following the discussion. I also used TRIZOL to extract RNA and have very low 260/280 spec. ratios. I have tried the Qiagen clean up and this has improved things. I wonder if I can use PCR amplification. That is; just thermo cycling with F &R primers no probes. Then run a TR PCR plate with the probes as a second step to get some sort of increase in 260/280 spec ratio. If so at what point do I make the cDNA?


Thank you

Sophia