Team:University College London/Protocols/week12/8

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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