RNA gel electrophoresis

Materials DEPC H2 O DEPC 0.1% (v/v) q.s. de-ioinized H2O 37ºC x1 hr, or r.t. overnight Autoclave. (NaOAc, EDTA and ethidium bromide solutions should also be DEPC treated.  Tris has a reactive amine and can''t be DEPC treated) 10x Formaldehyde gel loading buffer:  50% glycerol 10 mM EDTA, pH8 0.25% bromphenol blue 0.25% xylene cyanol 10x MOPS buffer (1L) 41.8 g MOPS (0.2M) in DEPC H2O,  pH7 20 mL 1 M NaOAC (DEPC treated) (final 20 mM) 20 mL 0.5M EDTA, pH8 (DEPC treated) (final 10 mM) q.s. 1L DEPC H2 O Sterile filter, store at 4ºC in dark.  (Don''t use if dark yellow) 1x Reaction buffer (per sample) 2 µL 10x MOPS buffer (final 1x) 4 µL formaldehyde (final 20%) 10 µL formamide (final 50%) 2 µL 0.2 mg/mL ethidium bromide, DEPC treated (final 10 µg/mL) 1.5% Agarose 2.2M formaldehyde gel 1.5 g agarose 72 mL H2 O Dissolve in microwave and then cool to 55ºC In a fume hood add: 10 mL 10x MOPS buffer 18 mL de-ionized formaldehyde Cast gel and wrap in Saran wrap until ready to use. Note: Northern blots require formadehyde in the gel.  For simple inspection, however, it is fine to use regular DNA-style TBE agarose gels.  In either case, the RNA should initially be denatured (steps 2-3) and RNAse free reagents should be used.   Procedure Add 2 µL RNA (up to 20 µg) to 18 µL 1x Reaction Buffer.  Incubate at 55ºC x1 hr, or 85ºC x 10 min.  Cool in ice and spin quickly to pull condensation down off of cap. Add 2 µL 10x Formaldehyde gel loading buffer. Load on agarose gel.  Use formaldehyde agarose gels and 1x MOPS running buffer if doing a Northern or if accurate sizes are necessary.  Otherwise 0.5x TBE gels are fine.  Rinse gel box in DEPC H2 O prior to use.  Use RNA standards.  Run at 4-5 V/cm for 4 hrs.  Place on Saran wrap prior to photographing