RNAgelelectrophoresis

實驗概要

RNA gel electrophoresis

主要試劑

DEPC H2O

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 H2O
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 H2O
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.

實驗步驟

1. 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.

2. Add 2 μL 10x Formaldehyde gel loading buffer.

3. 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 H2O prior to use.  Use RNA standards.  

4. Run at 4-5 V/cm for 4 hrs.  Place on Saran wrap prior to photographing

注意事項

         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.