Berglund:RNA column purification

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