Control of RNA

  1. From the RNA preparation take 5µl into a new 1.5ml tube.
  2. Add 995µl DEPC treated water.
  3. Adjust spectrophotometer to zero at 260nm and 280nm with quartz cuvette(s) filled with DEPC treated water from same batch as used to dilute RNA.
  4. Replace the water in the quartz cuvette in the measurement beam with RNA-solution.
  5. Measure absorbance at 260nm and 280nm.
  6. Multiply the reading at 260nm with 8 to get amount of RNA in µg/µl.
  7. Compare the readings at 260nm and 280nm the former should be approximately twice as high as the latter.

    RNA-gel

    Protocol for 100 ml gel - adjust amounts if necessary.

  1. Set water bath at 65°C.
  2. Mix 1.2g of agarose with 10ml 10x FA gel buffer and 90ml of DEPC treated water.
  3. Heat for 2-3 minutes in microwave oven until agarose is completely dissolved.
  4. Cool down to 65°C by placing in water bath at 65°C.
  5. Add 1.8ml 37% (12.3 M) formaldehyde and 1µl 10 mg/ml ethidium bromide.
  6. Mix and pour onto gel support from gel tank dedicated to RNA work, with masking tape at the ends.
  7. Insert comb and allow to set.
  8. Mix 55ml 10x FA gel buffer with 11ml 37% (12.3 M) formaldehyde and 484ml DEPC treated water to get 1x FA gel running buffer.
  9. Pour 550ml of 1x FA gel running buffer into gel tank dedicated to RNA work and submerge set gel.
  10. Allow gel to equilibrate for 30 minutes.
  11. To 5µl RNA preparation add 1.25µl 5x RNA loading buffer and mix.
  12. Incubate for 5 minutes at 65°C in water bath.
  13. Transfer immediately onto ice.
  14. After cooling give tubes a brief spin in centrifuge.
  15. Load onto equilibrated gel.
  16. Run gel for 1 to 3 hours at 100V until bromophenol blue is well down the gel.
  17. Inspect and photograph gel in UV-cabinet.

Solutions

DEPC treated water
To a bottle of dH2O add 1µl DEPC (diethyl pyrocarbonate) per ml of water, store overnight at 37°C, label with date and autoclave.

Gels

10x FA Gel buffer
Dissolve 83.72g of 3-[N-Morpholino]propanesulfonic acid (MOPS) (free acid) in 1800ml of DEPC treated water, add 33.33ml of sodium actetate pH 5.2 and 40ml 0.5M EDTA pH 8.0, adjust to pH 7.0 with NaOH, and adjust volume to 2l with DEPC treated water.
Store in cold room in bottles wrapped in tin foil.
5x RNA loading buffer
To 4.8µl saturated aqueous bromophenol blue solution add 8µl 500mM EDTA, pH 8.0, 72µl 37% (12.3 M) formaldehyde, 200µl 100% glycerol, 308.4µl formamide, 400µl 10x FA gel buffer, and 6.8µl DEPC treated water.
Can be stored in the fridge for up to 3 months.
Saturated aqueous bromophenol blue solution
Add solid bromophenol blue to DEPC treated water until there is solids left after vigorous mixing, spin down and use the saturated supernatant. Can be stored in the fridge.

Return