3 Purification of Nucleic Acids From Eukaryotic Cells
69
Note: For DEPC-treatment, see Subprotocol 5, Precautions; DEPC is suspected to be a carcinogen.
- Soak cuvettes for use with the spectrophotometer for 1h in concentrated H Cl:methanol ( 1:1) and then wash extensively with D EPC-treated water.
2. Suspend 0.5-1.0 g of oligo(dT)-cellulose in 0.1N NaOH. Pour a column
of oligo( dT)-cellulose (0.5-1.0 ml packed volume) in a sterile disposable
column or a pasteur pipet, plugged with sterile DEPC-treated and autoclaved glass wool.
Note: Up to 10 mg of total RNA can be loaded onto 1 ml of packed oligo( dT)cellulose. When using smaller quantities, the amount of oligo( dT)-cellulose
should be reduced accordingly to avoid lass of poly(A)+ RNA.
3. Equilibrate the oligo(dT)-column with 10-20 ml1 ( loading buffer, until
pH is <8.0.
4. Heat the RNA in water 5 min at 65°C. Cool quickly to room temperature, and add an equal volume of 2 x column-loading buffer.
Note: Heating the RNA disrupts regions of secondary structure that might
involve the poly(A)+ tail.
5. Apply the solution to the column, and immediatly begin to collect the
eluatein a sterile tube. Wash column with 1vol1 xcolumn-loading buffer and continue to collect.
6. Heat all the eluate to 65°C for 5 min and re-apply it to the column. Collect all eluate.
7. Wash the column with 5-10 column volumes of 1 x column-loading
buffer.
8. Elute the poly(A)+ RNA with 2-3 column volumes of elution buffer.
Collect fractions equivalent to 1/3 to 1/2 of the column volume.
Note: The oligo(dT)-column can now be stored at 4°C and reused many
times. Between uses, regenerate column by sequential washing with
NaOH, water, and 1 x column-loading bufferas described in steps 2-3.
9. Measure the absorbance of the fractions at 260 nm, using H Cl:methanol
treated cuvettes. Pool the fractions containg RNA.
10. Add 0.1 vol of 3M sodium acetate (pH 5.2). Mix by vortexing briefly or
by flicking the tube several times. Add 2.5 vol of ice-cold 100o/o ethanol,
mix and store for at least 30 min on ice.
Preparation of
poly(A)+RNA
69
Note: For DEPC-treatment, see Subprotocol 5, Precautions; DEPC is suspected to be a carcinogen.
- Soak cuvettes for use with the spectrophotometer for 1h in concentrated H Cl:methanol ( 1:1) and then wash extensively with D EPC-treated water.
2. Suspend 0.5-1.0 g of oligo(dT)-cellulose in 0.1N NaOH. Pour a column
of oligo( dT)-cellulose (0.5-1.0 ml packed volume) in a sterile disposable
column or a pasteur pipet, plugged with sterile DEPC-treated and autoclaved glass wool.
Note: Up to 10 mg of total RNA can be loaded onto 1 ml of packed oligo( dT)cellulose. When using smaller quantities, the amount of oligo( dT)-cellulose
should be reduced accordingly to avoid lass of poly(A)+ RNA.
3. Equilibrate the oligo(dT)-column with 10-20 ml1 ( loading buffer, until
pH is <8.0.
4. Heat the RNA in water 5 min at 65°C. Cool quickly to room temperature, and add an equal volume of 2 x column-loading buffer.
Note: Heating the RNA disrupts regions of secondary structure that might
involve the poly(A)+ tail.
5. Apply the solution to the column, and immediatly begin to collect the
eluatein a sterile tube. Wash column with 1vol1 xcolumn-loading buffer and continue to collect.
6. Heat all the eluate to 65°C for 5 min and re-apply it to the column. Collect all eluate.
7. Wash the column with 5-10 column volumes of 1 x column-loading
buffer.
8. Elute the poly(A)+ RNA with 2-3 column volumes of elution buffer.
Collect fractions equivalent to 1/3 to 1/2 of the column volume.
Note: The oligo(dT)-column can now be stored at 4°C and reused many
times. Between uses, regenerate column by sequential washing with
NaOH, water, and 1 x column-loading bufferas described in steps 2-3.
9. Measure the absorbance of the fractions at 260 nm, using H Cl:methanol
treated cuvettes. Pool the fractions containg RNA.
10. Add 0.1 vol of 3M sodium acetate (pH 5.2). Mix by vortexing briefly or
by flicking the tube several times. Add 2.5 vol of ice-cold 100o/o ethanol,
mix and store for at least 30 min on ice.
Preparation of
poly(A)+RNA