Top : Molecular Biology : DNA-Protein Interactions : Footprinting : Preparation of G+A Marker

Preparation of G+A Marker

Author: Long-Cheng Li
Source: Protocol Online
Date Added: Tue May 14 2002
Date Modified: Wed Apr 28 2004
Abstract: Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing reaction and works fine.

Procedure

  1. Add the following to a sterile microcentrifuge tube:
    Labeled target DNA (3-6ng)           1-8ul
    Calf Thymus DNA (0.5ug/ul)           2ul
    TE buffer                                   0-7ul      
    Total Volume                              10ul
  2. Add 1ul of 4% Formic Acid and incubate for 25 min at 37C
  3. During this incubation, add 15 ul of stock piperidine to 135 ul of water to prepare a 1M piperidine solution.
  4. Place the tube containing the formic acid reaction on ice, add all 150 ul of the diluted (1M) piperidine solution and incubate for 30 min at 90C.
  5. Place this reaction on ice for 5min, add 1ml of n-butanol and vortex.
  6. Centrifuge for 2mi8n at high speed to pellet the DNA. Remove the supernatant and add 150ul of 1% SDS to the pellet.
  7. Add 1ml of n-butanol, vortex vigorously and centrifuge at high speed for 2 min. Carefully remove the supernatant.
  8. Add 0.5 ml of n-butanol to rinse the pellet, and carefully remove the supernatant. Repeat this rinse step once.
  9. Dry the pellet under vacuum for 10min, adds 5-10ul of loading dye, and mix well. Place at ĘC20C until required. This sample may be stored up to two weeks at ĘC20C.

 Note

This protocol was adopted from Amersham footprinting kit instruction

(6/24/99)

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