IGEM:Harvard/2006/NES Notebook

To Order

• DMSO
• Sodium Thiosulfate
• Small, Medium, Large, X-large Nitrile gloves

Introductory Materials

IGEM:Harvard/2006/Lab_Intro

New Reporters

Due to issues with T7 promoter, the following biobricks were rehydrated 6/14

• J04430

Constitutive (IPTG-inducable?) GFP

• J04450

Constitutive (IPTG?) RFP

• I13522

Constitutive GFP using TetR promoter. No experience; might not work.

• I6060

Constitutive (IPTG?) YFP. Apparently gives uneven YFP expression

Transformed with 2ul of DNA on 15ul bacteria; twice for each plasmid. Took 3 ul from each incubation of and added into two two mixed SOC cultures. Let's see what happens when LacI GFP, RFP, YFP are co-cultured.

One of each LacI plasmid type, GFP, RFP, YFP, LacI were cultured overnight on plates treated with 10mM IPTG.

Microscope Notes

Overlap between FITC and YGFP blocks prevents differentiation between YFP and GFP. RFP can be differentiated between YFP/GFP very well.

YFP/GFP fades ~3 hours after being taken out of the incubator after overnight incubation. RFP was still strong at +3h.

IPTG doesn't make an apparent difference in fluoresence.

Promoter Research

7/25/06 Made Glycerol cultures of

• J04430 (lac GFP)
• J04450 (lac RFP)
• I13522 (tet GFP)

We need to transfer these constructs onto pSB4A3

Ligation Assay

• Digestions - Vary enzyme concentration

- 8 ul Midiprep Insert DNA + .5 ul each enzyme to 25ul -> 45C 6H

- 8 ul Midiprep Insert DNA + 1 ul each enzyme to 25ul -> 45C 6H

- 8 ul Midiprep Insert DNA + 1 ul each enzyme to 25ul -> 37C 6H

- 8 ul Midiprep Backbone DNA + .5 ul each enzyme to 25ul -> 45C 6H -> Phosphatase

- 8 ul Midiprep Backbone DNA + 1 ul each enzyme to 25ul -> 45C 6H -> Phosphatase

- 8 ul Midiprep Backbone DNA + 1 ul each enzyme to 25ul -> 37C 6H -> Phosphatase

• Run on Gel - Create purified and unpurified samples

Add 12 ul of each to the gel.

Chose the most successful digest and run a gel extraction.

• Ligation - Vary ratio of insert to backbone

Two insert samples:

- Gel purification of most successful digest

- Unpurified sample of most successful digest

Two backbone samples

- Gel purification of most successful digest

- Unpurified sample of most successful digest

For each combination create:

- 3ul backbone + 9 ul insert + ligase

- 1.5ul backbone + 15ul insert + Ligase

• Transform