Minnesota/18 July 2008

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|'''4. Ligate Digested products:'''  Ligated -  
|'''4. Ligate Digested products:'''  Ligated -  
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|a. Lac/LAMBDAcI promoter + GFP:Term + BaseVector
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|'''a.''' Lac/LAMBDAcI promoter + GFP:Term + BaseVector
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|b. TetR/p22mnt promoter + YFP + BaseVector
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|'''b.''' TetR/p22mnt promoter + YFP + BaseVector
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Revision as of 18:12, 21 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.
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