Fan Northern Blot Protocol

Part 1 RNA

1. You will want to load about 20ug of total RNA per lane. Furthermore, you can only load about 40ul of RNA + Loading Buffer in each well. Therefore, make sure your RNA is fairly concentrated (at least 2ug/ul)
2. Spec RNA on both the UV and on a gel. This will help you to load equal amounts in each well.

Part 2 Preparation of the gel

3. We use the small gel box specifically for RNA work (not one of the mini gels boxes). Make sure to clean and spray with 70% EtOH. If you feel the need, you can wash in DEPC water overnight.
4. I usually pour the gel in the gel box itself by turning the tray sideways. Also, make sure to use the fat side of the combs for the wells!
5. The gel we use is 1.2%. I have found that 120mLs is a good amount (enough that you can load ~40uL not to thick that you don't get a good transfer).
   1. 87mL of dH20
   2. 1.44g of Agarose
   3. 12mL of 10x MOPS
   4. 21ml of 37% Formaldehyde
6. Heat up agarose and dH20 first. Once in solution, let cool. Once cool enough add the 10x MOPS and the Formaldehyde in the hood! Mix and pour gel. Make sure to use the fat side of the comb! Also make sure there are no air bubbles in the gel.
7. Let cool in the hood

Part 3 More RNA prep

8. Mix RNA with loading buffer. Loading buffer consists of Formamide:Formaldehyde:10X MOPS in a 5:2:1 ratio. Thus, for loading, you want to mix the pre-made loading buffer with the RNA in a 4:1 ratio. For example, you would add 8ul of RNA to 32ul of Loading Buffer for a total of 40uL.
9. Heat RNA/Loading Buffer at 65° for 10 min and then cool on ice. Spin down
10. Add a bit of blue loading dye to the sample and load gel
11. Run at 80-100 volts until the bottom dye band is 2/3 of the way down.

Part 4 Transfer

12. After gel is finished running, you need to rinse it 4x for 15 min in dH20. This is to deionize and get rid of the formaldehyde in the gel.
13. After the 4th wash, place the gel in 20X SSC and let wash for 30min.
14. During this time, you can cut the paper towels, Whatman paper and Transfer Membrane to size.
15. Wash the Turbo Blotter with dH20 and then spray with 70% EtOH and dry.
16. Soak the transfer membrane in 20X SSC for 5 min
17. Pre-wet a few sheets of Whatman paper in 20X SSC
18. Set up the transfer in this fashion
    1. A big stack of paper towels on the bottom
    2. 4 ­ 5 sheets of dry 3M Whatman paper
    3. 1 pre ­ wet sheet of Whatman paper
    4. The transfer membrane ­ make sure there are no bubbles
    5. Place gel on top of this right side up ­ cut to size and make sure there are no air bubbles!
    6. 3 ­ 4 more sheets of 3M Whatman paper pre wet
    7. Place top of turbo blotter on and fill with transfer buffer (20X SSC)
    8. Add the pre-soaked wick ­ just a longer sheet of Whatman paper that covers the gel and reaches the wells of the blotter.
    9. Place some weight on top of the stack to help keep things pressed together ­ a 6 well plate and/or a pack of slides works well for this
    10. Cover the transfer apparatus with saranwrap or parafilm
    11. Let transfer overnight

Part 5 Post Transfer

19. Remove Whatman paper and take gel with membrane off. Make sure not to separate gel from membrane
20. Mark lanes with a pen and discard gel
21. Do a quick wash in 2X SSC ­ this is to help remove any pieces of agarose that might be stuck to the gel
22. Do a quick dry with paper towels and UV Crosslink on the optimal setting
23. After crosslinked, rinse blot in 1N Acetic Acid until the color of the dye changes
24. Pour Acetic Acid back in bottle
25. Next, wash in the 0.03% Methylene Blue solution ­ you should start to see staining of the 28S and 18S RNA on the gel
26. After sufficient color change, pour the Methylene Blue back into the bottle
27. Wash with dH20 until you see sharp bands for the 28 and 18S RNA
28. After washes, wrap blot in saran wrap and make a photocopy for records
29. Store unused blot in ­ 20°