Solutions
RNase-Free water
Prepare DEPC 0.1%(v/v) in Milli-Q water. Mix well, autoclave.
10X MOPS/EDTA
0.5M MOPS pH 7.0
0.01M EDTA pH 7.5 – 8.0
For 500mlL: 53.325g MOPS
10mL 0.5M EDTA pH 8.0
Bring up to 500mL with Milli-Q
pH to 7.0 with NaOH
Filter through 0.2μM filter
Store wrapped in aluminum foil
Formaldehyde/Formamide
89μL 37% formaldehyde
250μL deionized formamide
Buffer A
294μL 10X MOPS/EDTA
106μL RNase-free water
Gel Loading Buffer
322μL Buffer A
5mg xylene cyanol FF
5mg bromocresol green
400mg sucrose
Mix well, add:
178μL 37% formaldehyde
500μL formamide
Sample Preparation
1. Speed vac (on “medium” drying rate) 10μg total RNA, or 1-2μg poly A RNA. The lids of the tubes should be open, and their mouths covered with perforated parafilm.
2. Add 5μL Buffer A and resuspend RNA.
3. Add 12μL formaldehyde/formamide solution and mix.
4. Incubate samples at 68℃ for 10 minutes and place on ice.
5. Add 4μL gel loading buffer and mix.
Gel Preparation and Electrophoresis
1. Wash tray, comb and electrophoresis apparatus with 10% SDS and rinse thoroughly with Milli-Q. Alternatively, soak them in RNase free water overnight, or at least while performing the subsequent steps.
2. Prepare a 1% agarose gel in 1X MOPS/EDTA buffer and cool to 60℃.
3. In a fume hood add 9mL 37% formaldehyde to 41 ml of the cooled agarose. Mix and cast gel. Allow gel to polymerize for at least 45 minutes.
4. Pre-run gel at 40 volts for 45 minutes in 1X MOPS/EDTA buffer.
5. Load samples and run gel at 40 volts for small gels (80 volts for large gels).
6. Allow gel to run until bromophenol blue dye reaches ~ 1cm from the bottom.
