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352 Kösel et al.
Fig. 2. Sequencing results obtained with primers ND3 5′-TCC CCA CCA TCA TAG CCA-3′
and ND4 5′-GGG TTT TGC AGT CCT TAG-3′, which amplify a 302-bp segment of the
mitochondrial ND2 gene (3). Sequencing results were visualized using alkaline phosphataseconjugated
antibodies directed against digoxigenin and NBT/X-phosphate as an enzymatic
substrate. Contact blot of an 8% standard sequencing gel.
3. Purification of PCR products using the Wizard PCR Preps DNA Purification System: Add
50 to 100 µL of PCR product to 100 µL of purification buffer in an Eppendorf tube and
mix. Then, add 1 mL of purification resin and mix thoroughly. Pipet mixture into a 2-mL
syringe and push into a Wizard Prep column. Wash column with 2 mL of 80% isopropanol
and spin for 20 s in an Eppendorf centrifuge at full speed. Let isopropanol evaporate (takes
approx 5 min), and transfer column to new Eppendorf tube. Elute DNA by adding 50 µL
of 1× TE to the column. After 1 min, spin column for 20 s to recover DNA completely.
When ultrafiltration is used for purification of PCR products, 50 to 100 µL of PCR
product are diluted with approx 400 µL of double-distilled water and transferred to a
Microcon-30 concentrator. Centrifugation of the Microcon-30 column is done according
to manufacturer’s recommendation (e.g., 10 min at 12,000g) in an Eppendorf centrifuge.
Important: Avoid contamination of both types of columns with mineral oil used for
overlaying PCR.
4. A fluorometer is used to measure nanogram amounts of DNA. The TKO 100 (Hoefer) is
a useful and reasonably priced alternative to full-sized spectrophotometers for measuring
concentrations of double-stranded DNA. DNA concentrations as low as 10 ng/µL may be
reliably determined using a standard setup of the TKO. For measuring the concentration of
PCR products, the fluorometer is first calibrated with DNA standard (100 or 1000 ng/µL
calf thymus DNA, depending on the DNA concentrations to be determined): 1 µL of