互联网2013-11-13
Materials Supplied By User
1. Microcentrifuge capable of at least 10,000 x g.
2. Sterile 1.5 mL centrifuge tubes.
3. Sterile deionized water (or TE buffer)
4. Absolute (or 95%) ethanol
5. Protective eye-ware
1. Determine the volume of the labeling reaction, transfer to a clean 1.5 mL microfuge tube, and add 5 volumes of Buffer DP. Vortex thoroughly to mix.
NOTE: Radioisotopic labeling reactions yield sub microgram DNA due to the limiting nucleotide concentrations used. In this case, a carrier such as yeast tRNA should be added to Buffer DP to a final concentration of 2-5 ug/mL before use to ensure binding to HiBind resin. The mixture is stable at -70℃ for 2-3 months, but needs heating to redissolve salts in Buffer DP. Non-radioactive labeling reactions usually use higher dNTP concentrations and yield more product thus obviating the need for carrier.
2. Apply the sample to an HiBind™ DNA spin-column assembled in a clean 2 mL collection tube (provided) and centrifuge in a microcentrifuge at 10,000 x g for 1 min at room temperature. Discard the liquid.
4. Wash the column by adding 750 ul of Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000 x g for 1 min at room temperature.
Note: Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.
5. Optional: discard liquid and repeat step 5 with another 750 ul Wash Buffer.
6. Discard liquid and centrifuge the empty column for 1 min 10,000 x g to dry the column matrix. This is critical for good DNA yields.
7. Place column into a clean 1.5 mL microcentrifuge tube. Add 30-50 ul (depending on desired concentration of final product) sterile deionized water (or TE buffer) directly onto the column matrix and centrifuge 1 min at 10,000 x g to elute DNA. This represents approximately 80-90% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. Alternatively, elute a second time with the first eluate.
8. Yield and quality of DNA: determine the absorbance of an appropriate dilution of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows:
260 DNA concentration = Absorbance × 50 × (Dilution Factor) ug/mL
Probes greater than 500 bp in length can routinely be purified at >60% yield with greater than 95% removal of free nucleotides. Fragments ranging from 50 bp to 260 280 500 bp give yields of 30%-80%. The ratio of (absorbance )/(absorbance ) is an indication of nucleic acid purity. A value greater than 1.8 indicates > 90% nucleic acid. Activity of the probes will also need to be determined prior to setting up a hybridization reaction.
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