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RNA gel electrophoresis

Category: Experimental Procedures
Author: Admin eLabJournal

Labels: Electrophoresis, RNA

Materials

 

MOPS buffer 10X
Agarose
Formaldehyde (37%)
MOPS buffer 1X
Formamide
Ethidium bromide (100 mg ml-1)
Denaturation mix
Loading buffer: Orange G or Xylene cyanol/Bromophenol blue
Gel casting system
Gel running system
UV-transilluminator

Experiment settings

Volume of gel	405060708090100110120--other--ml
Agarose percentage	0.811.21.5--other--%

Step 1

--other--%  formaldehyde-agarose gel of --other--ml :

• --other--g  agarose
• --other--ml  10X MOPS buffer
• --other--ml  ddH₂O

Step 2

Dissolve in the microwave and allow to cool to 50°C

Step 3

Perform all next steps in the fumehood

Step 4

Add of --other--ml  of 37%-formaldehyde and add 1-2 µl of ethidiumbromide

Step 5

Swirl and pour the agarose-solution into the gel tray, place a comb with the appropriate number of wells and allow the gel to solidify

Step 6

Remove the comb, place the gel in a gel running system such that the gel is submerged in 1X MOPS buffer

Step 7

Denature 510--other--µg  of RNA in --other--µl  of denaturation mix for 15 min at 55°C.

Step 8

Mix the denatured RNA samples with 456--other--µl  of loading buffer and load the samples on gel

Step 9

Run the gel at 40 mA for 45 min and visualize the RNA in a UV-transilluminator.

• Make sure that all used materials are RNase free.
• Use gloves and change them regularly.

 

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