Transfer of nucleotic acids to membrane --- large fragment

1. For DNA fragments greater than 10 kb (e.g., plus field electrophoresis), transfer in a denature buffer (1.5 M NaCl, 0.25 M NaOH).

2. Deuprinate the DNA in 0.25 M HCl for 30 minutes at room temperature.

3. Denature in 1.5 M NaCl, 0.25 M NaOH for 30 minutes.

4. Transfer in 1.5 M NaCl, 0.25 M NaOH.

5. Wash the membrane in 1.5 M NaCl, 0.25 M NaOH.

6. Wash the membrane in 1 M NaCl, 0.5 M Tris-HCl pH 7.5.

See also Southern transfer