Preparation of Denaturing Agarose Gels

A variety of denaturants can be used with agarose. Alkaline gels are most often employed with single stranded DNA because pouring and handling such gels is not only nonhazardous, but convenient as well. However, because strong alkali will hydrolyze it, formaldehyde is used with RNA.

Preparing Alkaline Agarose Gels


At high temperatures, alkaline conditions will hydrolyze the Agarose polysaccharide chains. To prepare an alkaline gel, the agarose is first melted in water and cooled to near gelling temperature. Buffer concentrate is then added and the gel is poured.

GEL PREPARATION

  1. Dissolve 1.2g of agarose in 98ml of deionized water until all clumps are broken up. Heat the suspension in a microwave until it is a clear and homogeneous solution. Allow to cool to 15°C above gelling temperature.
  2. Add 2ml of 50X alkaline gel buffer (consisting of 1.5M NaOH and 50mM EDTA).
  3. Pour gel as described in the protocol on the previous page for the preparation of standard agarose gels.
  4. Run gel in 1X alkaline gel buffer.

SAMPLE PREPARATION:

Mix samples with an equal volume of 60mM NaOH, 2mM EDTA, 20% Ficoll and 0.06% Bromocresol Green. The stock solution consists of 2g Ficoll, 0.4ml 50X alkaline buffer, 6mg Bromocresol Green and enough deionized water to bring to 10 mL.



Preparing Formaldehyde Agarose Gels (for RNA Analysis)


Note: RNA is subject to rapid degradation by RNase present in the environment. For optimal results, use water treated with DEPC.

GEL PREPARATION:

  1. Melt 1.2g agarose in 87ml of DEPC water, by dispersing the agarose uniformly and heating in a microwave until all particles are dissolved.
  2. Bring the melted agarose to 60°C.
  3. Add 10ml 10X MOPS Buffer and 3ml 37% formaldehyde. Use DEPC treated water and RNase free reagents. FORMALDEHYDE IS VOLATILE AND TOXIC. WORK IN A HOOD FROM THIS POINT FORWARD.

    10X MOPS consists of the following (per liter):
    • 0.2M MOPS pH 7 with NaOH
    • 50mM sodium acetate
    • 10mM EDTA
       
  4. Pour gel as described in the protocol above for the preparation of standard agarose gels. USE A FUME HOOD!
  5. Allow gel to set for 1 hour.
  6. Run gel in 1X MOPS Buffer.

SAMPLE PREPARATION:

  1. Mix 40µl sample buffer with 10µl sample, heat to 55°C 15 minutes. The sample buffer is comprised of 65% formamide, 22% formalin (37% formaldehyde) and 13% 10X MOPS
  2. Add 10µl of the following: 50% glycerol, 1mM EDTA, 0.3% each bromophenol blue and 0.3% xylene cyanol.