Northern Blot
Samples:
Sample loading order – loaded µg unless otherwise indicated
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1. Prepare 1.2% agarose / formaldehyde gel:
-For ml:
a. Dissolve g agarose in (9/10 total volume) ml ddH20 and add ul EtBr
b. Cool to ~60C
c. In the hood add (1/20 total volume)ml formaldehyde and (1/20 total volume) ml 20x MOPS
d. Let gel solidify in hood.
2. Prepare ~ ml 1x MOPS running buffer.
3. Run gel for ~10min prior to loading
4. To prepare samples:
a. Move the amount of RNA solution containing (often 1-5ug)ug RNA to a new 1.5ml microfuge tube (See p. for calculations).
b. Dry the samples using the speed vac with heat at 37C
c. Add (typically 15ul)ul formaldehyde loading buffer to each sample and mix well.
5. Heat RNA at 65C for 5 min using a waterbath. Cool on ice prior to loading.
6. Load the entire samples onto the gel.
7. Run gel at (typically 100V)V for . Switch the direction of the leads (and the gel) every 30min to avoid exhausting the buffer.
8. View gel under UV light to assess RNA quality and loading.
9. Rinse gel in ddH2O and then 20x SSC for a combined total of ~10min.
10. Wet Hybond N+ Nylon Membrane (Amersham) cut to the size of the gel in 20x SSC. Do not force the membrane under the liquid.
11. Prepare transfer sandwich – no bubbles!
a. Cut 5 pieces of Whatman paper and several inches of paper towel to the size of the membrane.
b. Soaked 4 pieces of Whatman paper in 20x SSC and placed them on top of each other on a piece of plastic wrap.
c. Place the gel face down on Whatman paper. Use a serological pipette to gentle remove any bubbles (don’t squeeze).
d. Lay the membrane on the gel, remove bubbles, and frame with the edges of the plastic wrap.
e. Lay piece of Whatman paper soaked in 20x SSC on top of the membrane.
f. Add 2+ inches of paper towel and weigh down with a pyrex dish. Make sure everything is approximately level.
g. Let transfer overnight
12. Place damp membrane on a piece of paper towel and UV cross-link (Program C3 on the BioRad GS Gene Linker, total of 150mJoules).
-Note: At this point, can dry membrane, place between two pieces of Whatman paper, and store at 4C. Rewet with 20x SSC before continuing.
13. Warm hybridization buffer to in hybridization oven. (Based on Tm of hybrid; often 50-60C)
14. Place membrane gel side up in hybridization tube with 10-20ml of hybridization buffer. Incubate at for (minimum of 1hr).
CONDUCT ALL THE FOLLOWING STEPS IN THE RADIATION AREA.
15. If using newly synthesized probe, heat probe to 90C for 5min and cool on ice. Otherwise warm previously used hybridization buffer/probe solution to .
16. Remove hybridization buffer and add 10ml of hybridization buffer and probe.
17. Incubate at overnight with rotation.
18. Check self and work area for radioactive contamination.
19. Remove probe solution and store at -20C.
20. Wash membrane 3x 10 min with ~50ml of northern wash stringency 1 solution (2x SSC + 0.1% SDS).
-Note: All liquid goes in radioactive waste.
21. Dry membrane, wrap in plastic wrap, and expose to film at -80C.
22. Check self and work area for radioactive contamination.
Current as of 12-5-14