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Team:UESTC-China/Protocol
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Revision as of 04:44, 24 September 2014
DNA ligation with T4 DNA Ligase
| Component | 20ul Reaction |
|---|---|
| 1.33X Master Mix | 15ul |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| >Vector DNA | 50ng |
| ddH2O | To 20ul |
| Incubate at 50℃ for 1 hour |
DNA ligation with Gibson Assemble
| Component | 20ul Reaction |
|---|---|
| Vector DNA | 50ng |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| 10X T4 DNA ligase buffer | 2ul |
| T4 DNA ligase | 1ul |
| ddH2O | to 20ul |
| Incubate at 25℃ for 12 hours |
Double Digestion of DNA
| Component | 50ul Reaction |
|---|---|
| 10x FastDigest buffer | 5ul |
| Restriction enzyme 1 | 1ul |
| Restriction enzyme 2 | 1ul |
| DNA | 1-2ug |
| ddH2O | to 50ul |
| Incubate at 37℃ for 2 hours |
PCR protocol for KOD-Plus-Neo
| Component | 50ul Reaction |
|---|---|
| ddH2O | 33ul |
| 10X buffer for KOD-Plus-Neo | 5ul |
| MgSO4 | 1ul |
| 10mM dNTPs | 1ug |
| 10uM forward primer | 1ul |
| 10uM reverse primer | 1ul |
| Template | 1ul |
| KOD enzyme | 1ul |
