Using a Nylon Filter

Many groups now prefer the use of nylon filters, and varieties of charged (Hybond-N+) and noncharged nylon filters (Hybond-N) are commercially available. They have a high binding capacity for single-stranded nucleic acids; however, unlike nitrocellulose, nylon filters are capable of efficient double-stranded DNA retention. It is believed that nucleic acids bind to nitrocellulose in a noncovalent manner and baking will noncovalently bind nucleic acids to a nylon filter. Alternatively, a covalent association can be achieved by the UV irradiation of noncharged nylon filters and by simple alkaline treatment of positively charged filters. Further advantages of nylon-

based filters include their ability to bind smaller oligo-nucleotides and their enhanced structural strength permits secure long-term storage and further analysis such as multiple reprobing.

The techniques for blotting on to a nylon filter are similar to those for nitrocellulose supports; however, when using commercially acquired filters, it is recommended that manufacturers' instructions are followed. In general, when performing Southern transfers to a nylon membrane, alkali blotting is the method of preference. Compared to high-salt blotting, alkali blotting allows quicker transfer of DNA molecules (within in 2 hr) while minimizing the possibilities for DNA reassociation. Furthermore, on transfer to positively charged nylon filters, the DNA becomes covalently linked—thus removing the need for baking or UV treatment.

The method is fundamentally the same; however, no neutralization stage is necessary and the blotting apparatus only requires a few minor alterations from what was previously described (Fig. 1). The 20 x SSC transfer buffer is replaced with NaOH (0.4 mM) or NaOH (0.25 M) and NaCl (1.5 M) for positively charged and uncharged filters, respectively.1-4-1 Because of its hydrophilic nature, it is not necessary to prewet the nylon filter; it can be placed on the gel without any pretreatment and left for a minimum of 2 hr for DNA transfer—after which the apparatus are disassembled, the filter marked as described, probed, and stored for reference or further use.

Alkali-induced covalent linking of DNA to nylon filters works most efficiently for positively charged supports, and although it does work for uncharged nylon filters, UV treatment is the preferred method when using the latter.[4] However, overexposure to UV irradiation may reduce the ability of the nylon membrane to hybridize a probe[5] and this limitation is enhanced when using positively charged filters.[4]

Alkali blotting is not suitable for DNA transfer to nitrocellulose filters as these supports require a neutral pH to retain nucleic acids, and furthermore, their lack of structural stability is accentuated by alkali treatment. Performing Northern blots by alkali transfer is not recommended, because RNA is hydrolyzed by prolonged (overnight) alkaline exposure;[4] however, the use of lower (0.2 M) NaOH concentrations for shorter (1 hr) transfer times has generated successful Northern blotting techniques.1-6-1

Was this article helpful?

0 0
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment