Berglund:RNA column purification

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Have on hand before beginning: (By Amy)

transcribed RNA that has been DNased
filtered water
filtered 20% ethanol
Q column Buffer A: 10mM Tris pH 7.5, 50mM NaCl, 1mM EDTA (filtered)
Q column Buffer B: 10mM Tris pH 7.5, 1000mM NaCl, 1mM EDTA (filtered)
HiTrap Amersham FF Q column 1 ml with connectors and tubing
3cc luerlock syringe with needle

Method:
Pre-wash column (stored in 20% filtered Ethanol):
10ml of water
10ml of Buffer A
10ml of Buffer B
10ml of Buffer A

dilute 300µl Dnased RNA with 3ml of Buffer A
Load onto column with syringe

wash with:
100% Buffer A 3ml (discard)
30% Buffer B 3ml (0.9ml Buffer B + 2.1ml Buffer A)(these are free NTPs, discard)
100% Buffer A 3ml (discard)
70% Buffer B 3ml (2.10ml Buffer B + 0.9ml Buffer A)(collect; this should be your RNA) (I changed this on 5/26/09 because I realized I was loosing about 1/4 of my RNA with a 1.5ml volume)
100% Buffer B 3ml (discard, this is a wash to clean the column)
100% Buffer A 3ml (discard)

Wash with 3ml water
Wash with 3ml 20% ethanol

STORE COLUMN IN FRIDGE CAPPED SO IT CAN'T DRY OUT

Precipitate the RNA using final concentration of 0.3mM NaOAc pH 5.2 and 67% ethanol (3ml of RNA to 7.5ml of Ethanol and NaOAc)
Freeze in -20°C for 30min
Spin in JA17 at 14,500rpm (30,000rcf) for 10min

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