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Team:UESTC-China/Protocol
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Revision as of 15:10, 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 |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 68℃ | 30s/kb | ||
| Step3 | 68℃ | 5min | X1 cycles |
| 10℃ | 10min | ||
| 10uM reverse primer | 1ul | ddH2O | 33ul |
PCR protocol for KOD-Plus-Neo
| Component | 50ul Reaction |
|---|---|
| ddH2O | 32ul |
| 10X buffer for KOD-Plus-Neo | 5ul |
| MgSO4 | 3ul |
| 10mM dNTPs | 5ul |
| 10uM forward primer | 1ul |
| 10uM reverse primer | 1ul |
| Template | 1ul |
| KOD enzyme | 1ul |
| Each template will be diluted by the number of moles into 2-10ng/ul. |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 94℃ | 5min | X1 cycle |
| Step2 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 68℃ | 30s/kb | ||
| Step3 | 68℃ | 5min | X1 cycles |
| 10℃ | 10min |
Colony PCR with Taq DNA polymerase
| Component | 25ul Reaction |
|---|---|
| ddH2O | 15.8ul |
| 10X Taq buffer | 2.5ul |
| 2.mM dNTPs | 0.5ul |
| 10uM forward primer | 0.5ul |
| 10uM reverse primer | 0.5ul |
| Taq enzyme | 0.2ul |
| Water with colony | 5ul |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 95℃ | 5min | X1 cycles |
| Step2 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 72℃ | 1min/kb | ||
| Step3 | 72℃ | 10min | X1 cycles |
| 10℃ | 10min |
