Molecular Techniques and Methods

Formaldehyde Gel Electrophoresis for RNA Analysis

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The RNA is fully denatured, and its rate of migration through the gel containing formaldehyde is in linear proportion to the log10 of its molecular weight.




MATERIALS AND SOLUTIONS

10 X MOPS buffer (1 liter)
0.2 M MOPS ------------------------------------ 41.86 g
80 mM Na-acetate -------------------------------27 ml of 3 M Na-acetate
10 mM EDTA (pH8.0) -------------------------- 20 ml of 0.5 M EDTA
Add distilled H2O to make a final volume of ------ 1 liter
  • Adjust the pH to 7.0 with NaOH.
  • Filter through 0.4 um millipore membrane. Store in a dark bottle to protect from light.



  • Electrophoresis buffer (1 liter)
    10 X MOPS buffer ----------------------------- 100 ml
    37% Formaldehyde ----------------------------- 82 ml
    Distilled H2O ----------------------------------- 818 ml


    2x RNA Gel Loading Buffer (1 ml)
    50% Deionized Formamide ----------------------------- 500 ul of 100% Formamide
    12% Formaldehyde ------------------------------------ 334 ul of 37% Formaldehyde
    0.1% Xylene cyanol ---------------------------------- 20 ul of 5% Xylene cyanol
    0.1% Bromophenol blue ------------------------------- 20 ul of 5% Bromophenol blue
    1 mM EDTA ------------------------------------------- 2 ul of 0.5 M EDTA
    0.025% SDS ------------------------------------------ 2.5 ul of 10% SDS
    12.5% Glycerol -------------------------------------- 125 ul of 100% Glycerol


    Glycerol loading buffer (10 ml)
    50% Glycerol --------------------------------- 5 ml of 100% Glycerol
    1 mM EDTA(pH8.0) ------------------------- 20 ul of 0.5 M EDTA
    0.25% bromophenol blue --------------------- 1 ml of 2.5% bromophenol blue
    0/25% xylene cyanol -------------------------- 1 ml of 2.5% xylene cyanol
    Distilled H2O --------------------------------- 2.98 ml




    PROCEDURES

    Preparation of formaldehyde gel

    1. Melt by boiling 2 g of agarose in 146 ml deionized H20 plus 20 ml 10x MOPS Buffer.

    2. Cool the agarose to 65oC.

    3. In a fume hood, add 34 ml of a 37% Formaldehyde solution just prior to pouring the gel.

  • Formaldehyde is TOXIC. Work in a fume hood!
  • Do not add ethidium bromide in the gel. It is difficult to destain later.


    Preparation of RNA samples

    4. Prepare the sample by mixing the following in a sterile microfuge tube:

    RNA (up to 20 ug) ------------------------------------------ 10 ul
    2x RNA Gel Loading Buffer --------------------------------- 10 ul

    5. Denature the sample at 65oC for 10 min.

    6. Load the RNA samples onto the gel.

    7. Electrophoresis the RNA samples at 70 volts for 5-7 hrs.

    8. (Optional)

  • Stain the gel for 3-5 minutes in Ethidium bromide Staining Solution. Do not over-stain. It is difficult to destain later.
  • Destain the gel in 0.5x TBE for 30 min to overnight until bands appear.
  • Photograph the gel.
  • Detection limit >~25 ng/band.

    9. Continue the northern blot analysis.





    NOTES

  • Formaldehyde (M.W. = 30.03) is usually obtained as a 37% solution in water (12.3M).

  • Sample buffer should be discarded after use.




  • KIT INFORMATION




    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified