ELECTROPHORESIS OF RNA THROUGH FORALDEHYDE GELS

Formaldehyde is toxic and should be treated with care. At 0.1 ppm or higher concentrations in air, formaldehyde can irritate eyes and airways. Inhalation can cause headaches or a burning sensation to the throat, and can trigger asthma symptoms. The EPA classifies formaldehyde as a probably human carcinogen. Some countries ban the use of formaldehyde in certain consumer products, such as cosmetics. As a results, use dedicated formaldehyde glassware for all solutions containing formaldehyde. Similarly, gel trays are often dedicated to just formaldehyde work.

• 10X formaldehyde gel running buffer (10X MOPS).
• 0.2 M MOPS
• 10mM EDTA pH 8.0
• 10 mM Sodium Acetate pH 7.0 (adjust pH with 2 N NaOH and sterilize by filtration through 0.2 μm Nucleopore filters (Acrodiscs are probably fine)
• Deionize some formaldehyde (50 ml) and some formamide (20 ml) by stirring with amberlite on a stir-plate for 1 hour in the fume hood. Filter trough a Whatman #1 filter to remove the amberlite.
• Set water-bath at 55˚ C.
• 10X loading dye is:
  • 0.25% [w/v] xylene cyanol FF
  • 0.25% [w/v] bromophenol blue
  • 50 % glycerol
  • 10 mM EDTA pH 8.0
• Tank Buffer
  • Make 250 ml by diluting 10X MOPS 1:10 with DEPC treated DI water.

• Warm the formaldehyde and MOPS in the 55˚ C water bath.
• Add 0.3 g agarose to 18 ml DEPC treated DI.
• Microwave to melt the agar (your can do this in a 50 cc disposable tube). Make sure the agar is completely melted. DO NOT LET BOIL OVER!
• Place the molten agar into another beaker in the 55˚ C water-bath.
• Add 4.45 ml deionized fomaldehyde and 2.5 ml 10X MOPS to the 55˚ C molten agarose and mix well by swirling.
• Pour the gel in the fume hood and let harden for one hour.

• Add in a μfuge tube :

• 3 μl of your sample
• 5 μl deionized formamide,
• 2 μl deionized formaldehyde
• 1 to 1.5 μl 10X MOPS
• 1 μl of 0.4 mg/ml ethidium bromide.

• Vortex.
• Heat the samples in a PCR mashine to 65˚ C for 10 minutes.
• Cool samples on Ice.
• Prepare your size standard in a similar fashion.
• Add 1 μl of loading dye to each of the samples and standards and mix well.
• Preelectrophorese your gel for 5 min at 5V/cm (ca. 50 V).
• Immediately load samples and run at 3.5-4.5 V/cm for several hours.
• Half-way through the run unplug the rig, take your gel out and mix the tank buffer.