No discussions found
Start your discussion
Share your thoughts or question with experts in your field
Start a discussion
Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
|
After amplification, the PCR products were digested by the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique separately with the restriction enzymes, with recognition of the following sequence: 5′...CAG ↓ CTG...3′ and 5′...T ↓ CTAGA...3′, respectively for Pvull and Xbal in a final volume of 20 μl at 37 °C for 16 h, containing 6 U Pvull and 5 U XbaI (Promega), 2 μl enzyme buffer, 0.2 μl BSA (bovine serum albumin), and 17.3 μl PCR product. |
Fragment analysis was performed in 1% agarose gel stained with ethidium bromide, and the bands were separated and photographed under ultraviolet light. The PCR and RFLP conditions utilized followed the protocol of Souza et al. [16, 17]. |
| Protocol tips |
| After amplification, the PCR products were digested by the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique separately with the restriction enzymes, with recognition of the following sequence: 5′...CAG ↓ CTG...3′ and 5′...T ↓ CTAGA...3′, respectively for Pvull and Xbal in a final volume of 20 μl at 37 °C for 16 h, containing 6 U Pvull and 5 U XbaI (Promega), 2 μl enzyme buffer, 0.2 μl BSA (bovine serum albumin), and 17.3 μl PCR product. |
| Downstream tips |
| Fragment analysis was performed in 1% agarose gel stained with ethidium bromide, and the bands were separated and photographed under ultraviolet light. The PCR and RFLP conditions utilized followed the protocol of Souza et al. [16, 17]. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis. |
|
| Protocol tips |
| All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
All isolates were analyzed by using the restriction endonucleases BlnI (Takara) and XbaI (Takara). |
|
| Protocol tips |
| All isolates were analyzed by using the restriction endonucleases BlnI (Takara) and XbaI (Takara). |
Can't find the product you've used to perform this experiment? It would be great if you can help us by
Adding a product!