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Team:University College London/Protocols/week12/8
From 2012.igem.org
Ligations for nuclease, curli and laccase
Digestions:
PSB1C3 with E + P PCR amplified Laccase with E + P PCR amplified Curli with E+ P Nuclease in Puc57 with E + P
Constitutive promoter + RBS construct with E + S PCR amplified Laccase X + P PCR amplified Laccase X + P Nuclease in puc57 with X + P
Digestion protocol:
250ng vector: xuL Enzyme 1: 1uL Enzyme 2: 1uL NEB buffer 2: 2uL BSA: 0.5 uL dH2O: made up to 20uL
Incubated at 37C for 30mins and heat inactivated at 80C for 20mins.
The following ligations were then preformed:
Ligations were preformed using NEB quick ligase.
50ng of vector, 4uL, Combined in a (3:1) molar ratio with insert 10uL 2X Quick ligation buffer 1uL quick ligase
Incubate at room temperature at 5mins Transform immediately or store at -20
Nuclease positive cells
| Ligation Number | Components | Amounts |
|---|---|---|
| 1 | PCR amplified Curli | 12 ul |
| Constitutive promoter + RBS construct | 0.5ul | |
| PSB1C3 | 4.0ul | |
| 2 | PCR amplified Curli | |
| Constitutive promoter + RBS construct | 0.5ul | |
| PSB1C3 | 4.0ul | |
| 3 | Nuclease in puc57 | 2.5ul |
| Constitutive promoter + RBS construct | 0.5ul | |
| PSB1C3 | 4.0ul | |
| 4 | PSB1C3 | 4.0ul |
| PCR amplified Curli | 12 ul | |
| 5 | PSB1C3 | 4.0ul |
| PCR amplified Laccase | 6.0ul | |
| 6 | PSB1C3 | 4.0ul |
| Nuclease in puc57 | 2.5ul |
