Reagents:

Reduced Formaldehyde denaturing gel (1% Agarose, 1x MOPS, 1.9% di-F)

	20 ml	80 ml	100 ml
Agarose	0.2 g	0.8 g	1.2 g
25x MOPS	800 ul	3.2 ml	4.0 ml
H2O	18 ml	73 ml	91 ml
di-Formaldehyde	1.0 ml	4.0 ml	5.1 ml

25x MOPS (2 M MOPS, 500 mM NaOAc, 10 mM EDTA)ddH₂O

	50 ml	200 ml	400 ml
MOPS	20.91 g	83.65 g	167.3 g
NaOAc	3.40 g	13.6 g	27.2 g
EDTA	0.19 g	0.75 g	1.50 g
ddH2O	to 50 ml	to 200 ml	to 400 ml

Denaturing Buffer (1x MOPS, 50% Formamide, 2% Formaldehyde)

	150 µl	200 µl	750 µl	1500 µl
25x MOPS	6 ml	8 ml	30 ml	60 ml
di-Formamide	75 ml	100 ml	375 ml	750 ml
di-Formaldehyde	8 ml	11 ml	40 ml	80 ml
ddH2O	61 ml	81 ml	305 ml	610 ml

Blue Juice (50% Glycerol, 0.27% BPB/XC, 1.3 mM EDTA)

	1 ml	2 ml
Glycerol	0.5 ml	1.0 ml
Bromophenol Blue	2 mg	4 mg
Xylene Cyanol	2 mg	4 mg
0.5 M EDTA	2 ml	4 ml
ddH2O	498 ml	996 ml

Ethidium Bromide (1mg/ml)

� Blue Juice and Ethidium Bromide are added in 2:1 ratio.

� 100 ml gel: 50 µl sample (6 µl RNA + 30 µl denaturing buffer + 9 µl BJ/EB)

Protocol:

1. Dissolve agarose in MOPS and H₂O, cool to handwarm.

2. Add 37% di-Formaldehyde, poor gel, let solidify.

3. Flush wells after removal of comb.

4. Running buffer is 1x MOPS.

5. Add denaturing buffer to RNA samples in 5:1 ratio.

6. Incubate at 65°C for 15 minutes.

7. Cool on ice.

8. Add Blue Juice/Ethidium Bromide (2:1) to samples in 1:4 ratio.

9. Load on gel (max. 15 µl on 20ml gel, max. 50 µl on 100ml gel).

10. Run for 3 hrs at 75V.

Notes:

- Run 6 µg of RNA MW ladder along with samples.

- Optional: run ³²P labelled l/HindIII (10000 cpm) with samples.