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Wisconsin-Madison/20 June 2009
From 2009.igem.org
Ex 15: Inducing various Modified (Triple) E.Coli Strain Cultures 3
Used salt LB cannot continue. Re-innoculate with salt-free LB
Made liquid Cultures of E.Coli Strains in salt-free LB;
1. BL21 (DE3)
2. K-12 MG1655 AaraBAD
3. K-12 MG1655 (wildtype)
4. DH10B
Ex 16: Need to redo:
A. PCR clean up: Taq PCR Product - Promega Wizard Genomic DNA Purification Kit
B. Plan: Same sticky ends, ProU upstream of GFP:
Backbone: GFP: Cut with E and S – 32 mg/uL – 3720 bp
Insert: ProU: Cut with E and X – 120 mg/uL – 360 bp
100 x 5 x 360/ 3720 = 50
C. Digestion: 10uL rxn no water
D. Purification: according to Promega Wizard Genomic DNA Purification Kit
E. Ligation: 1 uL ligase
1 uL buffer
3 uL water
1 uL ProU
4 uL GFP - overnight
