Molecular Techniques and Methods

Alkali Blotting of DNA for Hybridization
(Southern Blotting Analysis)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

DNA fragments are separated on agarose gels and transferred from the gel to a nylon membrane using NaOH solution.




MATERIALS AND SOLUTIONS

Acid Depurination Solution (1 liter)
0.25 M HCl -------------------------------- 21.5 ml of 11.6 M HCl
Distilled H2O ------------------------------- 979.5 ml


Alkali Denaturation/ Blotting Buffer (1 liter)
0.4 M NaOH ------------------------------ 80 ml of 5 M NaOH
Distilled H2O ------------------------------- 920 ml




PROCEDURES

1. Electrophorese DNA samples in an agarose gel.
  • After electrophoresis, place the agarose gel in Acid Depurination Solution for 10~15 min.

    2. Rinse gel in distilled H2O.

    3. Fill tray with Alkali Denaturation/ Blotting Buffer.
  • Make a platform and cover it with a wick made from three sheets of Whatman 3MM filter paper, saturated with Alkali Denaturation/ Blotting Buffer.

    4. Place the gel on the wick and avoid trapping air bubbles beneath it.
  • Surround it with cling film to prevent the blotting buffer being absorbed directly into the paper towels above.

    5. Cut a sheet of nylon membrane to the exact size of the gel and place on top of the gel.
  • Avoid trapping bubbles beneath the membrane. If bubbles appear, they should be squeezed out using a glass rod or pipette.

    6. Place three sheets of 3MM paper, cut to size and wetted with
  • Alkali Denaturation/ Blotting Buffer, on top of the nylon membrane.

    7. Place a stack of absorbent towels (5 cm high) on top of the 3MM paper.

    8. Place a plate on top of the paper towels and put a 100 g weight on top.
  • Allow transfer to proceed for 2-24 hours.

    9. After blotting, dismantle the apparatus.
  • Before removing from the gel, mark the membrane with ball-point pen to allow later identification of tracks.

    10. Allow to air dry for up to an hour.

    11. Rinse the nylon membrane by immersion in 5 x SSPE or 5 x SSC before hybridization.

    12. Continue to hybridization.




    NOTES



    KIT INFORMATION



    REFERENCES


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