Preparation of G+A Marker

Author: Long-Cheng Li

Source: Protocol Online

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

Add the following to a sterile microcentrifuge tube:

Labeled target DNA (3-6ng)           1-8μl

Calf Thymus DNA (0.5ug/ul)           2μl

TE buffer                                   0-7μl     

Total Volume                              10μl

Add 1ul of 4% Formic Acid and incubate for 25 min at 37℃.

During this incubation, add 15 μl of stock piperidine to 135 μl of water to prepare a 1M piperidine solution.

Place the tube containing the formic acid reaction on ice, add all 150 μl of the diluted (1M) piperidine solution and incubate for 30 min at 90C.

Place this reaction on ice for 5min, add 1ml of n-butanol and vortex.

Centrifuge for 2mi8n at high speed to pellet the DNA. Remove the supernatant and add 150μl of 1% SDS to the pellet.

Add 1ml of n-butanol, vortex vigorously and centrifuge at high speed for 2 min. Carefully remove the supernatant.

Add 0.5 ml of n-butanol to rinse the pellet, and carefully remove the supernatant. Repeat this rinse step once.

Dry the pellet under vacuum for 10min, adds 5-10μl of loading dye, and mix well. Place at 20℃ until required. This sample may be stored up to two weeks at 20℃.

Note

This protocol was adopted from Amersham footprinting kit instruction(6/24/99)