Wisconsin-Madison/6 July 2009

July 6, 2009
'''Other:

Group Meeting

Received “ACTUAL” gsSDMT (Kanamycin Resistance)

Ex 10: Cyanobacteria

Inoculated new 6803 culture (20:1) in BG-11

Made freezer stock of the same

Ex 17: Salt Testing

Quick test of lac/GFP fusion, no fluorescence-maybe premature

Not correct OD for testing (OD600 = 0.3)

Ex 14: Obtaining gsSDMT and

'''Ex 19: Parts from Registry

Last gene we has was not correct, now have actual gsSDMT

1. Transformed gssdmt CESG gene into DH10B

SEE ABOUT GENE FOLDER

2. Transformed GFP with RBS lac/GFP fusion from kit plate



Ex 19: Parts from the Resgistry

Miniprep of Lac and RFP

Digestion of Lac and RFP

On Gel in next listed Experiment

Ex 20: New Cloning Methods

A. Double Digestion:

-	Lac (E+S)

-	GFP (E+X)

-	NudF (X+P) – PCR Product

-	Choline (X+P) – PCR Product

-	Backbone (X+P) (blunt, Scott’s Protocol)

B. SAP Treatment: of backbone to prevent backbone re-ligation

VSAP treatment –30 min - Clean-up (Magnetic Beads)

50 uL	 Digest

6 uL 	10x Buffer (scott)

3 uL 	water

1 uL	SAP (Scott)

(60uL) - Cleanup

C. Ran Gel:

Lac1 cut, lac1 uncut, Lac2 cut, Lac2 uncut, RFP1 cut, RFP1 uncut, RFP2 cut, RFP 2 uncut, Ladder, F, C, BB Lac	- bp

RFP	- bp

NudF	560 bp

PEAM 2000 bp

BB 	2100 bp

D. Spec:

NudF (560)	46 ug/mL	(3)

PEAM (2000)28 ug/mL	(17)

BB (2100)	28 ug/mL

E. Ligation

Insert: 100 x (insert size bp/backbone size bp) x 5 uL

F. Thermocycler: users: Dan Agnew: Lig - overnight