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Found 4 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
|
Approximately 3 μg PCR products in 250 μl of
1× CutSmart buffer was mixed with 50 U BbsI-HF,
20 U rSAP, and incubated at 37 °C for 2 h, followed
by deactivation of rSAP at 80 °C for 20 min. MluCI
(20 U) was subsequently added to the mixture and
the digestion was continued at 37 °C for 1 h. The
resulting mixture was extracted once with
phenol/chloroform/isoamyl alcohol (25:24:1, v/v)
and the aqueous phase was evaporated, desalted by Waters Oasis HLB extraction cartridges (Milford,
MA), and redissolved in 10 μl water. |
|
| Protocol tips |
Approximately 3 μg PCR products in 250 μl of
1× CutSmart buffer was mixed with 50 U BbsI-HF,
20 U rSAP, and incubated at 37 °C for 2 h, followed
by deactivation of rSAP at 80 °C for 20 min. MluCI
(20 U) was subsequently added to the mixture and
the digestion was continued at 37 °C for 1 h. The
resulting mixture was extracted once with
phenol/chloroform/isoamyl alcohol (25:24:1, v/v)
and the aqueous phase was evaporated, desalted by Waters Oasis HLB extraction cartridges (Milford,
MA), and redissolved in 10 μl water. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
A modified protocol using the FastDigest versions (Thermo Fisher Scientific, USA) of the BsaI and BpiI restriction enzymes were used with an initial 10 min digestion, 20–50 cycles of digestion and ligation (37 °C for 2 min, 16 °C for 5 min), followed by a final digestion step and a heat inactivation step, was used for most assemblies, instead of the standard MoClo protocol. |
|
| Protocol tips |
| A modified protocol using the FastDigest versions (Thermo Fisher Scientific, USA) of the BsaI and BpiI restriction enzymes were used with an initial 10 min digestion, 20–50 cycles of digestion and ligation (37 °C for 2 min, 16 °C for 5 min), followed by a final digestion step and a heat inactivation step, was used for most assemblies, instead of the standard MoClo protocol. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
Set up a BbsI digest by mixing in an Eppendorf tube 300 μg plasmid DNA (see Note 9), 30 μL BbsI (150 U), and 60 μL 10× NEB2, and add H2O to 600 μL total volume. Mix well (without vortexing) and incubate at 37 °C overnight in a Thermomixer. |
|
| Protocol tips |
| Set up a BbsI digest by mixing in an Eppendorf tube 300 μg plasmid DNA (see Note 9), 30 μL BbsI (150 U), and 60 μL 10× NEB2, and add H2O to 600 μL total volume. Mix well (without vortexing) and incubate at 37 °C overnight in a Thermomixer. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
LRP6 CRISPR-Cas9 targeting construct was generated by ligating the annealing product of the primers listed in Supplementary Table 1 to pX330 (obtained from Addgene #48139) digested with BbsI (Thermo Scientific). |
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| Protocol tips |
| LRP6 CRISPR-Cas9 targeting construct was generated by ligating the annealing product of the primers listed in Supplementary Table 1 to pX330 (obtained from Addgene #48139) digested with BbsI (Thermo Scientific). |
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