INTRODUCTION
This protocol is for hybridization between 100% matched sequences.
For hybridization between heterologous sequences (less than 90% matches), use low stringent condition.
MATERIALS AND SOLUTIONS 10 x MOPS Buffer (500 ml)
200 mM MOPSpH 7.0 --------------------------------- 21 g
80 mM Sodium acetate -------------------------------- 13.5 ml of 3 M Sodium acetate
10 mM EDTA ----------------------------------------- 10 ml of 0.5 M EDTA
Add DEPC-trated H2O to make a final volume of --- 500 ml
Electrophoresis buffer (1 liter) 10 X MOPS buffer ------------------------------------ 100 ml 37% Formaldehyde ------------------------------------ 82 ml
Distilled H2O ------------------------------------------- 818 ml
Formaldehyde interacts with O2 to become formic acid. Minimize air-exposure.
2x 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
Ethidium bromide Staining Solution (100 ml)
100 ug Ethidium bromide ------------------------------- 10 ul of Ethidium bromide Stock Solution (10 mg/ml)
100 mM Ammonium acetate ------------------------- 1 ml of 10 M Ammonium acetate
DEPC-trated H2O ------------------------------------- 99 ml
Desolve until background fluorescence is minimal.
Keep in aluminum foil-covered bottle.
Prehybridization Solution (20 ml)
50% Deionized Formamide ----------------------- 10 ml of Deionized Formamide
25 mM PIPESpH 6.8 ----------------------------- 0.5 ml of 1 M PIPES
5 mM EDTA ---------------------------------------- 0.2 ml of 0.5 M EDTA
10 X Denhardt's solution -------------------------- 2 ml of 100 X Denhardt's solution
0.75 M NaCl ---------------------------------------- 3 ml of 5 M NaCl
1% SDS ------------------------------------------- 2 ml 10% SDS
2 mg Salmon Sperm DNA ------------------------ 0.2 ml of Salmon Sperm DNA (10 mg/ml)
DEPC-trated H2O -------------------------------- 2 ml
PROCEDURES
Gel Preparation
1. Prepare formaldehyde agarose gel as follow.
Target mRNA > 2kb
0.8%
Agarose
0.48 g
DEPC-H2O
44 ml
10x MOPS Buffer
6 ml
Target mRNA < 2kb
1%
Agarose
0.6 g
DEPC-H2O
44 ml
10x MOPS Buffer
6 ml
2. Cool the agarose to 65oC.
3. In a fume hood, add 10 ml of a 37% Formaldehyde solution just prior to pouring the gel.
Formaldehyde is TOXIC. Work in a fume hood!
Denaturation of RNA
4. Mix 10-20 ug total RNA (or, 1-2 ug poly A+-RNA) with equal volume of 2x Gel Loadling BUffer.
5. Denature RNA for 5 minutes at 90oC.
6. Cool down RNA on ice.
This step prevents the secondary structure formation.
Electrophoresis and Blotting
7. Load the RNA sample in the gel and electrophorese the RNA in Electrophoresis buffer at 4-5 V/cm until the BPB migrated 8 cm (4-5 hours) in a fume hood.
Optional: After electrophoresis, stain the gel for 10 minutes in Ethidium bromide Staining Solution. Photograph the gel.
Optional: If the RNA to be analyzed is >2.5kb in length, treat the gel with alkaline solution to partially hydrolyze RNA.
Caution, too much treatment can degrade RNA completely.
(a) Soak the gel for 5 min in 300 ml (5 gel vol) of 0.05N NaOH.
(b) Soak the gel for 30 min in 600 ml (10 gel vol) of 20x SSC.
For heterologous sequence hybridization, use low stringent condition.
Ex) Increase NaCl concentration to 1 M in prehybridization solution.
Decrease hybrization temperature to 30-35oC.
Wash membrane at room temperature.