3 Purification of Nucleic Acids From Eukaryotic Cells
67
9. Carefully remove the ethanol.
10. Add 1 ml 70o/o ethanol to remove precipitated salt, vortex briefly, and
incubate 10-15 min at room temperature.
11. Repeat steps 8 and 9.
12. Dry pellet in a desiccator under vacuum or in a Speedvac® evaporator
for 5-15 min, and dissolve in 100-200 111 DEPC-treated water.
13. Quantitate the nucleic acid content by spectrophotometric determination of an aliquot at wave-lengths of 260 nm and 280 nm.
Subprotocol 5
Preparation of Poly(A)+ RNA
Principles of poly(A)+ RNA preparation. The protocols for preparing RNA
from eucaryotic cells yields total RNA, which contains primarily tRNA and
rRNA. Since the vast majority of mRNAs in mammalian cells carry tracts of
poly(A) at their 3' -termini, they can be separated from the bulk of cellular
RNA by their capacity to bind to oligo(dT)-cellulose (Edmonds et al. 1971;
Aviv and Leder 1972). This step is essential for construction of cDNA libraries and usually improves Northern hybridization or nuclease-S1 assays
(Sambrock et al. 1989).
Materials
• 0.1M NaOH
• 3 M sodium acetate (pH 5.2)
• lOo/o (w/v) sodium lauryl sarcosinate
• 1 x column buffer
• 2 x column loading buffer
• elution buffer
• 70o/o and lOOo/o ice-cold ethanol
• RNAse-free TE-buffer (pH 7.0) (10 mM Tris-HCl, pH7.0, 1 mM EDTA
(pH 8.0))
Reagents