Minnesota/18 July 2008

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1. Analyze gel results from 07-17-2008: Send in select samples from the gel for sequencing. Send in reaction mixture containing template DNA and primers that will bind to it, and thus allow to sequence. Sequencing L3i, L3j, L4b, L4i, L5c, L6a, L6b, L6d, L6e, L7a, L7b, L7d, and L8a.

2. Problem: No growth on plates with MCherry, RFP, Terminator, and TetR promoter.

Solution: Re-transform (again) paper DNA with MCherry, RFP, Term., and TetR.

3. Double Restriction Digest: Double digest dually repressed promoters; (1) Lac/LAMBDAcI, (2) TetR/p22mnt, along with reporter genes; GFP and YFP. Incubate @ 37C for 2-20 hrs. Heat inactivate for 15 mins @ 65C. Follow the table below:

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2
(A) Lac/LAMBDAcI Promoters	5.0uL	0.5uL	13.5uL	29.0uL	1.0uL, EcoRI	1.0uL, Spe1
(B) Lac/LAMBDAcI Promoters	5.0uL	0.5uL	9.5uL	33.0uL	1.0uL, EcoRI	1.0uL, Spe1
(A) TetR/p22mnt Promoters	5.0uL	0.5uL	34.1uL	8.4uL	1.0uL, EcoRI	1.0uL, Spe1
(B) TetR/p22mnt Promoters	5.0uL	0.5uL	34.1uL	8.4uL	1.0uL, EcoRI	1.0uL, Spe1
GFP:Term	5.0uL	0.5uL	20.5uL	22.0uL	1.0uL, EcoRI	1.0uL, Xba1

RFP will not be used because it has not been properly transformed yet. Will result in:

Lac/LAMBDAcI:GFP:Terminator

Tet/p22mnt:YFP:Terminator

4. Ligate Digested products: Ligated -

a. Lac/LAMBDAcI promoter + GFP:Term + BaseVector

b. TetR/p22mnt promoter + YFP + BaseVector

Parts	10x Buffer	H20	Prom	Reporter	T4 Ligase
(a) Lac/LAMBDAcI : GFP : Term	3.0uL	6.0uL	15.0uL	5.0uL	1.0uL
(b) Lac/LAMBDAcI : GFP : Term	3.0uL	6.0uL	15.0uL	5.0uL	1.0uL
TetR/p22mnt : YFP	3.0uL	6.0uL	15.0uL	5.0uL	1.0uL

5. Work on model: Model allows us to run potential reactions computationally. This will give us an idea of whether or not there will be errors in the rxns and what outputs will result.