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Found 4 matching solutions for this experiment
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Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). |
The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). |
| Protocol tips |
| Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). |
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| The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). |
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Genomic DNA (500 ng) was digested with 50 U of HpaII, HhaI, MspI (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers. The digested DNA was recovered by standard ethanol precipitation and dissolved in 10 μl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Note that the amount of DNA used for digestion is dependent on the number of amplicons to be tested. For smaller amount of DNA, use of appropriate carriers would be helpful to ensure efficient recovery. |
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| Protocol tips |
| Genomic DNA (500 ng) was digested with 50 U of HpaII, HhaI, MspI (TaKaRa), or McrBC (New England Biolabs) overnight at 37°C in 50 μl of the recommended buffers. The digested DNA was recovered by standard ethanol precipitation and dissolved in 10 μl of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Note that the amount of DNA used for digestion is dependent on the number of amplicons to be tested. For smaller amount of DNA, use of appropriate carriers would be helpful to ensure efficient recovery. |
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RFLP typing used the enzyme HinP1I (NEB): 6 μl of the PCR product was added to 0.4 μl 10× NEB buffer 2, 0.3 μl HinP1I (NEB), and 3.3 μl H2O and then digested at 37°C for 4 h. |
A single representative clone of each RFLP type was amplified for sequencing using the protocol above and substituting Expand high-fidelity polymerase (Roche) for Taq DNA polymerase. |
| Protocol tips |
| RFLP typing used the enzyme HinP1I (NEB): 6 μl of the PCR product was added to 0.4 μl 10× NEB buffer 2, 0.3 μl HinP1I (NEB), and 3.3 μl H2O and then digested at 37°C for 4 h. |
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| A single representative clone of each RFLP type was amplified for sequencing using the protocol above and substituting Expand high-fidelity polymerase (Roche) for Taq DNA polymerase. |
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5-15 μl of the PCR products were digested with BsuRI, Hin6I, and HphI (Thermo Scientific) in 20 μl volumes. The samples were incubated at 37°C for 1-2 hours, and the enzymes were then inactivated with ten-minute incubation at 80°C. The digests were analyzed on 2.5–3% agarose gel. |
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| Protocol tips |
| 5-15 μl of the PCR products were digested with BsuRI, Hin6I, and HphI (Thermo Scientific) in 20 μl volumes. The samples were incubated at 37°C for 1-2 hours, and the enzymes were then inactivated with ten-minute incubation at 80°C. The digests were analyzed on 2.5–3% agarose gel. |
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