Biochemical Techniques and Instrumentation

Southern Blot Protocol

1. Digest genomic DNA at 37 ° C overnight.
   1. 10μg DNA with 5 Units restriction enzyme / 1μg DNA
   2. Final volume 50μL
2. Inactivate enzyme by incubating at 65 ° C for 15 min.
3. Add 10μL 6x loading buffer.
4. Load sample on a 0.7% TBE agarose gel (400 mL size).
5. Use 10μL 1 kb plus DNA ladder.
6. Run gel overnight at 60V.
7. Stain gel:
   1. Put gel in tupperware.
   2. Pour ~1 L TBE running buffer from gel box into tupperware.
   3. Add 50 m L ethidium bromide to TBE.
   4. Shake on orbital shaker at 24-26 rpm for ~30 min.
8. Make picture of gel—take fluorescent ruler along.
9. Treat gel with 1 L of 0.25 M HCl (20.8 mL 12 M HCl in 979.2 mL ddH2O) for 20 min. During incubation time, bromophenol blue will turn yellow (pH indicator).
   • For higher molecular weight fragments it may be better to use 0.4 M HCl for up to 30 min.
10. Rinse the gel with dH2O.
11. Treat gel with Denaturing Solution (DS) twice for 20 min. each. Use 1 L each treatment:
    Denaturing Solution:
    • 1.5 M NaCl (330 mL 5 M NaCl)
    • 0.5 M NaOH (50 mL 10 M NaOH)
    • 620 mL dH2O
12. Equilibrate gel for 5-15 min. in Transfer Buffer (TB). Use 1 L:
    Transfer Buffer:
    • 1.5 M NaCl (330 mL 5 M NaCl)
    • 0.25 M NaOH (25 mL 10 M NaOH)
    • 645 mL dH2O
13. Setting up the blot (air bubbles are your enemy!):
    1. Lay a TB-prewetted double layer of Whatman paper (fits size of the gel) onto the transfer tray, it should reach the buffer reservoir on both sides.
    2. Put the gel flipped upside-down onto the Whatman paper, remove air bubbles from between the Whatman paper and the gel by rolling gently with a 5 or 10 mL serological pipet.
    3. Stretch parafilm across three sides of tray surrounding the gel to prevent paper from wicking anywhere else but through the gel. Parafilm should prevent paper towels, etc from touching reservoir.
    4. Put a TB-prewetted nylon membrane (Hybond N+, Amersham) on the gel.
    5. Put 3 layers of TB-prewetted Whatman paper on top of it.
    6. Put 1 dry Whatman paper on top.
    7. Put 3 layers of blotting papers (Sigma) on top of Whatman papers.
    8. Pour ~1 L Transfer Buffer into tray.
    9. Stretch parafilm over remaining side of tray.
    10. Add ~10-20 cm layers or 2/3 of a pack of paper towels (the white ones are the better soakers).
    11. Put horizontal surface on top of paper towels. Make sure the entire thing is level.
    12. Put a weight on top (an empty bottle works well). It may be useful to cover the whole blotting tower with plastic wrap to prevent drying out (if run over the weekend).
    13. The transfer time should be 12-24 hours (O/N).
    14. After transfer, rinse the membrane in 2X SSC with gentle shaking for a few minutes and allow to dry completely (30-45 min).
    15. UV cross-link and store between 2 sheets of Whatman paper.