Double-stranded DNA Denaturation Buffer, also known as Neutral Transfer Buffer, is primarily composed of 1.5 M NaCl, trace NaOH, and other components, and does not contain sucrose. This reagent is specifically designed for nucleic acid hybridization experiments, particularly suitable for the denaturation of double-stranded DNA in Southern blot experiments.
This product is for research use only. Not for use in diagnostic procedures or other purposes.
Procedure (For Reference Only)
The product can be used directly according to specific experimental requirements. The following are dedicated steps for DNA denaturation and transfer for different membrane types:
1. Transfer to Uncharged Membranes
1.1 DNA Denaturation: Place the gel in 10 volumes of Double-stranded DNA Denaturation Buffer. Incubate at room temperature for 45 minutes with continuous gentle agitation.
1.2 Neutralization Treatment: Briefly rinse the gel with deionized water. Subsequently, submerge the gel in 10 volumes of DNA Neutralization Buffer I. Incubate at room temperature for 30 minutes with gentle agitation. Replace with fresh DNA Neutralization Buffer I and continue incubation for another 15 minutes.
2. Transfer to Charged Nylon Membranes
2.1 Alkaline Denaturation: Place the gel in 5-10 volumes of DNA Alkaline Transfer Buffer. Incubate at room temperature for 15 minutes with continuous gentle agitation.
2.2 Secondary Denaturation: Replace with fresh DNA Alkaline Transfer Buffer and continue incubation for another 20 minutes with gentle agitation.
Notes
If the target DNA fragment length is less than 20 kb, it is recommended to avoid performing depurination reactions to prevent fragment degradation.
If the single-use volume is small, it is recommended to aliquot the reagent appropriately before use to minimize the impact of repeated freezing and thawing on reagent stability.
Use the reagent as soon as possible after opening to avoid reagent inactivation or contamination, which could affect subsequent nucleic acid transfer and hybridization experiment results.
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