PROTOCOL FOR NORTHERN BLOTTING

Developed by: Christopher Todd Hittinger
Modified from: "Bio-Rad Vacuum Blotter Instruction Manual" and "Analysis of RNA by Northern and Slot Blotting Hybridization" from Short Protocols in molecular Biology

Preparation

1. EVERYTHING must be Thoroughly cleaned of RNAses, and uLoves must be worn at all times.

2. Use RNAse-ZAP or some other commercially availABLe RNAse cleaner to clean the work area and all equipment that may come into contact, directly or indirectly, with the RNA. If none are available, use a dilute solution of bleach.

3. Clean everything with 95 % ethanol. This will also help any residual RNAse-ZAP evaporate.

4. Put sign(s) around the work area that stress(es) the importance of a lack of contamination.

5. Also, use only DEPC-treated (diethylpyrocarbonate) chemicals.

6. Chemicals can be DEPC-treated by adding 2 mL of DEPC per L of solution, followed by autoclaving.

Pouring the Formaldehyde Gel (for a 1 % gel)

7. Put 0.50 g agarose into an Erlenmeyer flask.

8. Add 36 mL of DEPC-H2O.

9. Add-5 mL of DEPC-10×MOPS.

10. Add 9 mL of 12.3 M (37 %) formaldehyde (does not need DEPC-treatment).

11. Boil in microwave to dissolve agarose. BE CAREFUL to inhale as little of the formaldehyde fumes as possible.

12. Pour gel into set up box with comb in it. If using 66 p-L loading samples as described here, 3 wells must be taped together to form I using the 8-welled comb in order for the sample to fit. For any sized loading,- sample, DO NOT use the outer wells.

LoADIng the Gel (66 uL sample described, but ratios are what is important)

16. Add 27.5 mL of formamide.

17. Incubate in a PCR machine for 15 minutes at 55° C