RNA gel electrophoresis
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1730
Materials
DEPC H2 O
1x Reaction buffer (per sample)
Procedure
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: 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)
50% glycerol
10 mM EDTA, pH8
0.25% bromphenol blue
0.25% xylene cyanol
10x MOPS buffer (1L)10 mM EDTA, pH8
0.25% bromphenol blue
0.25% xylene cyanol
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)
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 gel4 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 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.
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.
- 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
