1 2 3 4 5 6

5. Work on absorbent paper. Add 0.2 ml chloroform to each tube. Cap tubes carefully. Shake tubes by hand vigorously
for 15 sec. and store at room temp. for 2-3 minutes. Centrifuge at 11,500 rpm for 15 minutes.

6. Transfer clear aqueous (top) phase to clean labeled microfuge tube. Do nottransfer any of the interphase containing
DNA (cloudy) or the organic phase containing proteins (pink). Add 0.5 ml isopropanol. Mix by gentle inversion.
Incubate samples at room temperature for 10 min. Centrifuge at 11,500 rpm for 10 minutes.

7. Discard supernatant by gently pouring off.
Add 1 ml 75% ethanol. Mix and store at -80ºC.

Quantitation of RNA
1. Spin down chick embryo total RNA at 9,500 rpm for 5 min.
Carefully pour off supernatant and invert tube on clean kimwipe.
Allow to air dry for 10 minutes.

2. Resuspend in 40 ul sterile dH2O (containing RNase inhibitor).
[optional: If your pellet was small, only resuspend in 20 ul.]
Gently pipet up and down with sterile tip, and incubate briefly at 55ºC.

3. Dilute 5 ul into 1 ml (1/200 dilution).
Measure absorbance (A) at 260 and 280 nm using the spectrophotometer.
The A260 can be used to estimate the concentration; a 40 ug/ml solution of RNA will have an absorbance of 1. The
A260/A280 ratio gives an estimate of purity.


A260 (dil)

Total OD


Yield (ug)

5 ug=(ul)


4. Calculate the concentration of your RNA [A260 x 200 (dilution factor) x 40 ug/ml].
Transfer 5 ug to a sterile tube for RNA gel analysis, and 10 ug to a second tube containing 1 ul oligo-d(T) primerfor
cDNA synthesis (keep on ice). Do not use more than 16 ul. Store remaining RNA at -70ºC.

@2004 Cebra-Thomas

Last Modified: 17 August 2004

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