| 1. Plasmid DNA Purification Using the QIAprep: Use 2mL LB cultures of ligation products from 07-11-2008. Procedure:
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| a. Resuspend bacterial cells in 250uL Buffer P1 and transfer to microcentrifuge tube
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| b. Add 250 uL Buffer P2 and invert 4-6 times
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| c. Add 350uL Buffer N3 and mix immediately by inverting 4-6 times
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| d. Centrifuge for 10 minutes @ 13,000 rpm in a table-top centrifuge. White pellet will form (made up of cell debris).
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| e. Apply supernatants from step D to the QIA prep spin columns. Centrifuge for 30-60 seconds --> discard the flow through.
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| f. Wash the QIA prep spin column by adding 0.5mL (500uL) Buffer PB. Centrifuge for 30-60 seconds. Discard the flow through.
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| g. Wash QIA prep spin column by adding 0.75mL (750uL) Buffer PE. Centrifuge for 30-60 seconds. Discard the flow through.
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| h. Centrifuge for additional 1 minute to remove residual buffer.
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| i. Place the QIA prep spin column in a clean 1.5mL centrifuge tube. To elute DNA, add 50uL of Buffer EB (10mM Tris-Cl, pH 8.5) to center of spin column and centrifuge.
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| NOTE: Used Buffers to purify or washout any cell debris so only has DNA. Used spin column b/c the column binds to DNA (has DNA affinity) and all other solution will drain through.
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| 2. Spectrophotometry: 'Spec' purified preps to check concentration of DNA in the ligated products. Refer Spectrophotometry Results link on Notebook page.
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| 3. Double Digest: Perform double digest. Incubate @ 37C for 2-20hrs. Heat inactivate enzyme @ 65C for 15 mins. Vector Dephosphorylation next; Base Vector only.
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| GFP + Terminator => GFP:Term.
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| Pro/LAMBDAcI + Terminator => Pro:LAMBDAcI:Term.
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| Pro + LAMBDAcI/Terminator => Pro:LAMBDAcI:Term.
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| Refer to Double Digest table below.
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2
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| GFP (1250 ng/uL) | 5.0uL | 0.5uL | 41.5uL | 1.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| Terminator | 5.0uL | 0.5uL | 17.5uL | 25.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Promoter/LAMBDAcI | 5.0uL | 0.5uL | 40.5uL | 2.0uL (L3b) | 1.0uL, Pst1 | 1.0uL, Spe1
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| Promoter/LAMBDAcI | 5.0uL | 0.5uL | 32.5uL | 10.0uL (L3c) | 1.0uL, Pst1 | 1.0uL, Spe1
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| LAMBDAcI/Terminator | 5.0uL | 0.5uL | 27.1uL | 15.4uL (L4b) | 1.0uL, EcoRI | 1.0uL, Xba1
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| LAMBDAcI/Terminator | 5.0uL | 0.5uL | 20.3uL | 22.2uL (L4c) | 1.0uL, EcoRI | 1.0uL, Xba1
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| BaseVector (35 ng/uL) | 5.0uL | 0.5uL | 13.5uL | 29.0uL | 1.0uL, EcoRI | 1.0uL, Pst1
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| TetR Promoter | 5.0uL | 0.5uL | 8.5uL | 34.0uL | 1.0, EcoRI | 1.0uL, Spe1
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