Minnesota/24 July 2008

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|'''3. Base Vector & Promoter Double Digest:''' 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps:
|'''3. Base Vector & Promoter Double Digest:''' 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps:
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!| Parts !! 10x Buffer !! BSA !! H20 !! DNA from parts !! RE 1 !! RE 2 !! [DNA] ng/uL
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| BV  || 5.0uL || 0.5uL || 30.8uL || 11.7uL || EcoRI, 1.0uL || Pst1, 1.0uL || 100.62 ng/uL
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| TetR Pro. 1 || 5.0uL || 0.5uL || 35.5uL || 7.0uL || EcoRI, 1.0uL || Pst1, 1.0uL || 23.8 ng/uL
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| Tet R Pro. 2 || 5.0uL || 0.5uL || 35.5uL || 7.0uL || EcoRI, 1.0uL || Pst1, 1.0uL || 23.8 ng/uL
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| LacI Pro. 1 || 5.0uL || 0.5uL || 41.5uL || 1.0uL || EcoRI, 1.0uL || Pst1, 1.0uL || 168.0 ng/uL
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| LacI Pro. 2 || 5.0uL || 0.5uL || 41.5uL || 1.0uL || EcoRI, 1.0uL || Pst1, 1.0uL || 168.0 ng/uL
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| Lac/Lam 1 || 5.0uL || 0.5uL || 28.5uL || 14.0uL || EcoRI, 1.0uL || Pst1, 1.0uL || 9.8 ng/uL
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| Tet/p22 1 || 5.0uL || 0.5uL || 34.0uL || 8.5uL || EcoRI, 1.0uL || Pst1, 1.0uL || 20.4 ng/uL
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| Tet/p22 2 || 5.0uL || 0.5uL || 34.0uL || 8.5uL || EcoRI, 1.0uL || Pst1, 1.0uL || 20.4 ng/uL
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|'''4. Vector Dephosphorylation:'''

Revision as of 16:18, 24 July 2008

Back to Notebook Home
Go to Previous Day (July 23)Go to Next Day (July 25)
1. Base Vector Plasmid Maxi Prep Base Vector: Grew another culture of different base vector colonies, so now need to plasmid prep the base vector so the DNA/plasmid will be extracted from the rest of the cell.
2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA.
3. Base Vector & Promoter Double Digest: 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps:
Parts 10x Buffer BSA H20 DNA from parts RE 1 RE 2 [DNA] ng/uL
BV 5.0uL 0.5uL 30.8uL 11.7uL EcoRI, 1.0uL Pst1, 1.0uL 100.62 ng/uL
TetR Pro. 1 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL
Tet R Pro. 2 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL
LacI Pro. 1 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL
LacI Pro. 2 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL
Lac/Lam 1 5.0uL 0.5uL 28.5uL 14.0uL EcoRI, 1.0uL Pst1, 1.0uL 9.8 ng/uL
Tet/p22 1 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL
Tet/p22 2 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL
4. Vector Dephosphorylation:
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