Southern Blot

yujian623

Southern Blot
Day 1
1. Run a big gel. Load 50 ml of each sample. Make sure to use blue loading dye. Use 120V. It may take about 4 hours.
2. Check gel under UV light and take picture with fluorescence ruler.
Measure size of the gel. Cut one corner (lower left) off as the marker of the orientaton.
3. Put gel in 0.25N HCl (20.6 ml/L). Shake slowly until the blue marker changes to greenish yellow and then shake ten more minutes.
4. While waiting, prepare paper towels, filter paper and membrane.
Unfold and cut paper towels to the same size as the gel.
Cut 6 pieces of Whatman filter paper to the same size as the gel.
Cut a piece of Nucleic Acid transfer membrane (Amersham) to the same size as the gel. Mark the side, that faces the gel. Cut a corner (lower left) off the membrane. Soak the membrane in water.
Cut two long pieces of Whatman filter paper with the same width of the gel to serve as a bridge.
5. Place gel in 0.4N NaOH (32g in 2L).
6. Prepare bridge. Put two layers of filter paper over the gel box. Roll with half a pipet to get rid of bubbles. Fill both ends of gel box with 0.4N NaOH.
7. Carefully place gel upside down (the cut corner on lower right) on the bridge. Try to put it in the right place at the first time. Place the top of gel (where DNA is) close to upper end of bridge, near the buffer. Roll out air bubbles.
8. Place membrane on top of the gel. It should be the same size. If the gel is a little bigger, trim the extra area with a razor blade. Roll out air bubbles.
9. Wet 3 layers of filter paper in 0.4N NaOH, place them on top of membrane and roll out air bubbles. Add 3 layers of dry filter paper, then add stock of paper towels (2-3 inch thick). Fill the chamber with 0.4N NaOH.
10.  Place gel tray on top of paper towels and balance with level. Add weight on top gel tray and use tapes to hold the gel tray in place. Transfer DNA to the membrane overnight.
Day 2:
1. Turn hybridization oven on at 65°C.
2. Prepare wash solutions
A. 6x SSC
60ml (20x SSC) + H2O