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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.
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