Nucleic Acid Dot/Slot Blotting

with MAGNACHARGE Nylon Membrane

Sample Preparation

Purify DNA or RNA by cell lysis or by suspended methods in 50 µl of TE buffer/pH 8.0. For RNA, adjust the pH to 7.5.

DNA:
Denature DNA with the addition of 0.1 vol of 3 M NaOH. Incubate for 1 hour at 60°C. Cool and add 1 vol 2 M NH₄OAc/pH 7.0, or 6 x SSC.

RNA:
Denature RNA by adding 35 µl of 20 x SSC, and 20 µl of 37% formaldehyde. Incubate for 15 minutes at 60°C.

Membrane Preparation

Wet membrane in DI water, then soak in the appropriate buffer.

DNA - Soak in 1 M NH₄OAc/pH 7.0, or 6 x SSC.

RNA - Soak in 20 x SSC

Sample Application

If using a filtration manifold, cut 2 pieces of filter paper to the size of the manifold plate and soak them in:

DNA - 6 x SSC

RNA - 20 x SSC

Place filter paper, then membrane onto the sample plate and secure the dot blotting or slot blotting unit. Apply low vacuum, and wash the wells with either 500 µl of 1 M NH₄OAc/pH 7.0, or 6 x SSC for DNA, 500 µl of 20 x SSC for RNA. Apply sample (not to exceed 10 µg DNA or RNA/well), under low vacuum. Remove membrane. If working with RNA, rinse each well again with 500 µl of 20 x SSC.

If dotting directly onto the membrane, apply 2-5 µl samples to membrane placed on 2 sheets of dry filter paper. Allow to dry.

Immobilization

Immobilize by baking for one hour at 80°C or bake for 30 minutes at 80°C and expose the membrane to a controlled UV source at 200 µj (x100), or to a transilluminator, at a wavelength of 302 nm for 30 seconds. Increased exposure can cause a decrease in signal intensity proportional to the amount of overexposure.

Hybridization

See prehybridization, hybridization, and post-hybridization procedures within the Southern and Northern Hybridization section.

Detection

Detect with isotopic or colorimetric probes. Nylon membranes can be scanned by laser densitometry.

Please call GE Osmonics' Labstore Support at (800) 444-8212, by fax (952) 988-6662 or email us for questions, pricing and availability. For international support and distributor locations, call +1-952-988-6665 or email us.