| 1. Maxiprep the 2 50.0mL cultures of Base Vector: Maxi-prep (just like the QIA prep but with larger amount of solution) the base vector cultures.
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| 2. Spec the base vector products: Using spectrophotometry, find the concentration of DNA in the base vectors grown O/N. Results: 0% concentration of DNA. We are screwed.
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| 3. Kat takes control: Kat says that we should pick different base vector colonies again and grow larger amounts of them O/N while incubating. Tomorrow these new cultures will be maxi prepped using the expensive prep kit, and then spec'ed to see if there is any DNA concentration within the base vector plasmids.
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| 4. Sarah writes out a plausible schedule: Sarah designated a flexible schedule -
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| a. Double Digest BV and Tet R promoter. BV will use restriction enzymes EcoRI & Pst1; TetR will use restriction enzymes EcoRI & Pst1.
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| b. Vector Dephosphorylate
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| c. Gel Purify: Separate BV remains from TetR promoter, also separate BV from ccdB gene.
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| d. Ligate BV & TetR Promoter ***PICTURE***
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| e. Transform pSB3K5:TetR
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| f. Double Digest TetR promoter & LAMBDAcI. TetR will use restriction enzymes Spe1 & Pst1; LAMBDAcI will use r.e.'s Xba1 & Pst1.
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| g. Vector Dephosphorylate
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| h. Gel Purify
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| i. Ligate TetR promoter & LAMBDAcI. ***PICTURE***
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| j. Transform pSB3K5:TetR:LAMBDAcI
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| k. Double digest TetRp:LAMBDAcI & Terminator. The Terminator will use restriction enzymes Xba1 & Pst1; whereas TetRp:LAMBDAcI will use r.e.'s Spe1 & Pst1.
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| l. Vector Dephosphorylate
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| m. Gel Purify
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| n. Ligate TetRp:LAMBDAcI & Terminator. ****PICTURE****
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| o. Transform.
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| NOTE: Must repeat all steps for all 3/4 parts
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