Formaldehyde denaturing agarose gel electrophoresis

Summary

Agarose gel electrophoresis is commonly used to isolate RNA prior to Northern blotting.

Operation method

Formaldehyde denaturing agarose gel electrophoresis

Materials and Instruments

10XMOPS Electrophoresis Buffer Formaldehyde Formamide 10X Sampling Buffer Ethidium Bromide Agarose DEPC Treated Water
Horizontal electrophoresis unit Water bath

Move

(i) Materials and equipment

1) Horizontal electrophoresis unit

2) Water bath

3) 10XMOPS electrophoresis buffer: 0.2 mol/LMOPS (pH 7.0), 20 mmol/L sodium acetate, 10 mmol/LEDTA (pH 8.0)

4) Formaldehyde; 37% solution (13.3 mol/L)

5) Formamide (deionized): Mix 10 ml of formamide with lg of ion exchange resin, stir for lh and filter through Whatman filter paper, aliquot into 1 ml and store at 70 °C.

6) 10X Sampling Buffer: 10 mmol/LEDTA (pH 8.0), 0,25% bromophenol blue, 0.25% xylene cyanide, 50% glycerol.

7) Ethidium bromide

8) Agarose

9) DEPC treated water


(ii) Method of operation

1) Gel preparation: 100 ml. of agarose gel was prepared, containing no 2.2mol/L formaldehyde and 1I.5% agarose. Dissolve 1.5 g of agarose in 72 ml of water plants, microwave to dissolve and cool to 55X:, add 10 ml of 10XMOPS electrophoresis buffer and 18 mL of deionized formaldehyde, and then fill the gel.

2) Sample preparation; measure spectrophotometrically to determine concentration. If RNA concentration is too low (<lmg/ml), concentrate by precipitation with ethanol or isopropanol.

3) Electrophoresis: Denaturation reaction in a l.5 mL centrifuge tube with the following components.

RNA (up to 20ug) 2ul

10XMOPS 2ul

Formaldehyde 4ul

Formamide 10ul

Ethidium bromide (200ug/ml) 1ul

After mixing, hold at 55℃ for 60 min to denature the RNA, cool in ice for 110 min, centrifuge, add 2ul10X sampling buffer and mix well, put on ice, electrophoresis, use 1XMOPS as electrophoresis buffer until the bromophenol blue reaches the bottom of the gel. The results were visualized under UV light.

4) Molecular quality labeling of RNA. ①Use the size standard of commercial RNA molecules. ②Use the two main ribosomal RNAs in the cell: 18S (about 2.lkb) and 28S (about 4.5kb) as molecular mass reference.

Caveat

1) For high resolution. You can add more methanol to the gel. Electrophoresis at a lower current, e.g. 20mA, overnight. Run the gel in a cold room or use a circulation pump to prevent overheating, but this is not necessary.2) Staining of the samples can also be done after electrophoresis by staining the gel in ethidium bromide solution (0.5ug/ml) for 10 min. If the background is too high the gel can be decolorized in aqueous plants for 30 min.3) Formaldehyde-containing agarose gels are more brittle than normal gels and should be handled with care.4) On formaldehyde-containing agarose gels, RNA migrates faster than DNA of equal length, so the size of unknown molecules should not be measured with a standard molecular mass reference for DNA.5) The electrophoresis tank for RNA electrophoresis should be washed with detergent solution, rinsed with water, dried with ethanol and then filled with 3% hydrogen peroxide, and then left at room temperature for 10 min before rinsing the tank thoroughly with DEPC-treated water.

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