| 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 to Spectrophotometry Results link on Notebook page.
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| 3. Double Digest: Perform double digest. The double digest was performed using the reaction mixtures as shown in the table below. The appropriate restriction enzymes were determined based on the information provided in Engineering BioBrick vectors from BioBrick parts. GFP, one of our two reporter genes, was cut on either end using EcoR1 and Spe1, since it will be the 'prefix part' in its subsequent ligation to the terminator biobrick. As the suffix, the terminator was cut using Xba1 and Pst1. The product from 7/11 were digested such that a third insert could be ligated on either end. The Promoter/Lambda cI construct was cut using Spe1 and Pst1. This will open the base vector, allowing the terminator to be 'slid into place.' Similarly, the Lambda cI/Terminator construct was cut using EcoR1 and Xba1, allowing for insertion of the promoter. Incubate @ 37C for 2-20hrs. Heat inactivate enzyme @ 65C for 15 mins.
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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 the Double Digest reaction 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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| 4. Vector Dephosphorylation : The 'vector' component for each of ligation reaction was vector dephosphorylated. This should prevent the vector from self-ligating without the insert. Refer to Dephosphorylation performed on 07-11-2008 for details.
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| 5. Meeting with Yiannis: Yiannis states that we need to: (1) Start preparing for presentation to be given on the last Wednesday of internship, (2) Use Emma's old report/paper as a guide or starter on writing own paper that is due at end of internship, (3) submit parts to iGEM, look @ Yiannis' email and start to work on what iGEM requires and what would iGEM want for medals, (4) slightly change model or change reactions to get RFP or GFP since it is slightly mixed up, (5) PUT LINK ON WIKI FOR SYNBIOSS.
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| 6. Ligation: The ligation of our recently digested parts will allow them to rejoin ends, with an additional insert, as described above. After ligation is finished, leave @ room temperature to ligate for 1 hour. Refer to the Ligation Reaction table below:
| Part(s) | 10x Buffer | H20 | BV | Insert DNA 1 | Insert DNA 2 | T4 Ligase | #
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| BV/GFP/Term | 3.0uL | 8.0uL | 2.0uL | 4.0uL GFP | 12.0uL Term | 1.0uL | L5
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| 2x Pro:LAMBDAcI/Term | 3.0uL | 8.5uL | 0 | 2.5uL Pro:LAMBDAcI | 15.0uL Term | 1.0uL | L6, L7
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| 2x Pro/LAMBDAcI:Term | 3.0uL | 8.5uL | 0 | 15.0uL Pro | 2.5uL LAMBDAcI:Term | 1.0uL | L8, L9
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| NOTE: 2x refers to 2 samples being used. BV refers to Base Vector. Pro refers to promoter. Term refers to terminator.
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| 7. Transformations: Transform ligated products to DH5alpha competent E. Coli cells. Follow previously performed transformation steps for details. Place transformations in 1.0mL cultures of 2xTy broth; place cultures in incubator @ 37C with shaking @220 rpm's for 2 hours. Plate ligated cell cultures on kanamycin resistant plates; allow to grow overnight @ room temperature.
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