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Northern Blotting 1. Measure gel to set up transfer. After run, cut off bottom right corner of gel. To remove
 

Summary: 1
Northern Blotting
1. Measure gel to set up transfer. After run, cut off bottom right corner of gel. To remove
formaldehyde, rinse gel at least 3 X 10 minutes in dH2O. Glyoxal gels require no rinsing.
2. If gel is >5 mm thick, or is >1% agarose, or the RNA to be analyzed is >2.5 kb, then:
A. Soak gel for 15 minutes in freshly made 50 mM NaOH at room temperature.
B. Rinse gel in dH2O.
C. Soak gel 2 X 15 minutes in 20X SSC at room temperature prior to transfer.
3. Cut membrane (e.g., nitrocellulose, nylon) ~1 mm smaller than gel. Wet in dH2O. Soak 5 minutes
in 20X SSC.
4. Cut 2 sheets of thin filter paper and 2 sheets of thick filter paper. Wet filter paper in 20X SSC.
5. Cut 2-3 inch stack of paper towels about the same size as the nitrocellulose.
6. Set up transfer as shown below. Cut 2 long pieces of thin filter paper to make wick. Place wick
over glass plate on support platform (e.g., plastic tip box). Wet wick with 20X SSC. Remove air
bubbles at this and all steps during set up. Put gel face down on wick. Place nitrocellulose on gel.
Add thin and thick filter papers. Add 50-100 g weight on top (glass plates work well). Place
plastic wrap around gel and wick to prevent evaporation.
7. After transfer, wash membrane 5 minutes in 2X SSC. UV cross-link (see below).
Gel
Thick

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine