Wisconsin-Madison/20 June 2009

June 20, 2009
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