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Document: mRNA isolation using Oligo - dT columns | Last modified: December 28, 2005
Isolation Of Poly A+ RNA From RNA Using Oligo-dT Column Chromatography
Xiaoping Yang 10-18-98

Take care that everything is RNase free and wear gloves.
Use disposable items from unopened stock or "RNA only" stock.
All glassware must be rinsed with DEPC water and baked prior to use.
All solutions made with DEPC water and chemicals used are for RNA only

Treatment of total RNA

Total RNA is dissolved in DEPC water with eppendorf tube
Add 50 ul of 10 M LiCl per ml sample.
Heat RNA at 70 deg C for 10 min, chilled on ice 5min just before loading on column.

Column preparation

— Use a disposable 5ml syringe.
— Remove the plunger and put in a pinch of glass wool, enough to stop the Oligo-dT from passing out of the syringe.
— Push glass wool down to the bottom using the plunger.
— Wash the column with 5ml of 10N NaOH, and DEPC water 5ml X 3. (This is necessary to avoid oligo-dT sticking to the top of the column)

— Weigh out 0.2 g oligo-dT Cellulose [Pharmacia, type 7] into the syringe. [approximately 0.6 cm in height]. A general rule is to use 25 mg of oligo(dT) for each 1mg of total RNA.

— Wash with 5ml of 0.1 M NaOH
— Wash with 10 ml DEPC H20.
— Equilibrate with 20 ml loading buffer (LB).
— Test pH of eluant by paper. Should be 7.5.

Chromatogarphy

— Label the columns with sample number. One column, one sample.
— Load samples and collect flow thru.
— Reload the first flow thru and collect flow thru again. Repeat again.
— Save total RNA in DEPC.H20 at -20 deg C in case all poly A+ does not bind.

— Wash column with 15-20 ml MWB.
— Collect last 5 ml in 1 ml aliquots.
— Measure OD at 260nm of these fractions. Should be down to about 0.1 and stable. If not, wash column until OD 260 is down to 100 ug poly A

Regeneration of column

— Wash with 10 ml EB
— 10 ml DEPC water
— 5 ml 0.1N NaOH
— 20 ml DEPC water
— 20 ml 100% ethanol
— Cap and store at -20 deg C.

Buffers

LB (loading buffer)
0.5 M LiCl
10 mM Tris (pH=7.5)
1 mM EDTA
0.1% SDS
Adjust final volume to 500ml with DEPC water

MWB (middle wash buffer)
0.15 M LiCl
Rest as LB

EB (elution buffer)
2.mM EDTA
0.1% SDS
Adjust final volume to 500ml with DEPC water

DEPC water
Add 2 ml DEPC to 1L DDW. Put @ 37 deg C overnight.
Autoclave with caps loosened.
Store at RT
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