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
Sample buffer (1 ml)
10 X MOPS buffer ----------------------------100 ul
37% Formaldehyde(12.3 M) ------------------ 175 ul
Fresh frozen formamide ------------------------ 500 ul
Glycerol loading buffer (below) ---------------- 220 ul
10 mg/ml EtBr --------------------------------- 5 ul
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. Prepare the gel by melting 1.5 g agarose in 108 ml deionized
H2O. Cool to 60oC.
2. In the hood, add the followings to the agarose solution from step 1.
10 X MOPS buffer ---------------------------- 15 ml
37% Formaldehyde (12.3 M) ------------------ 27 ml
3. Prerun the formaldehyde gel for 10 min at 70 volts.
Preparation of RNA samples
4. Prepare the sample by mixing the following in a sterile microfuge
tube:
RNA (up to 20 ug) ---------------------------- 5 ul
Sample buffer --------------------------------- 9 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. 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