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 m 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.

Department of Microbiology
School of Medicine
University of Washington
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