Minnesota/24 July 2008

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 * 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, which was 430 ng/uL.
 * 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:
 * 2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA, which was 430 ng/uL.
 * 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:


 * 4. Vector Dephosphorylation: After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below:
 * 4. Vector Dephosphorylation: After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below:


 * 5. Gel electrophoresis: After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.
 * 6. Gel Purified: Cut out bands from gel and purified those bands (which contain the DNA wanted). Follow QIA protocol: (1) Excise the DNA fragment from the agarose gel with a clean blade, (2) Place gel fragments in designated 2.0mL tubes, (3) Add 1.0mL of Buffer QG to gel in 2.0mL tubes - allow to solubilize (dissolve) for 10 minutes @ 50C and mix by vortexing, (4) After the gel slice has dissolved completely add 1.0mL of 100% isopropanol to the samples and mix, (5) Place samples into a QIAquick spin column to bind DNA and centrifuge for 1 minute, (6) Discard flow through and place spin column back in same collection tube, (7) Add 0.5mL of Buffer QG to spin column and centrifuge for 1 minute, (8) Discard flow through and add 0.75mL of Buffer PE to spin column and centrifuge for 1 minute, (9) discard the flow through and centrifuge for additional 1 minute @ 10,000rcf, (10) Place column into a clean 1.5mL microcentrifuge tube, (11) To elute DNA add 50uL of Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of QIAquick membrane and centrifuge for 1 minute. The flow through is the purified DNA, which will be used in spectrophotometry.
 * 7. Spetrophotometry: Spec the purified gel DNA to measure concentration of DNA in eight parts. 9 wells have 36.0uL of distilled water, the 1st well will have 4.0uL of buffer EB added as the control, the following 8 wells will have 4.0uL of the designated DNA from the purified gel.
 * 8. Ligations: Ligate the spec'ed purified DNA samples. Follow the table below:
 * 7. Spetrophotometry: Spec the purified gel DNA to measure concentration of DNA in eight parts. 9 wells have 36.0uL of distilled water, the 1st well will have 4.0uL of buffer EB added as the control, the following 8 wells will have 4.0uL of the designated DNA from the purified gel.
 * 8. Ligations: Ligate the spec'ed purified DNA samples. Follow the table below:
 * 8. Ligations: Ligate the spec'ed purified DNA samples. Follow the table below:
 * 8. Ligations: Ligate the spec'ed purified DNA samples. Follow the table below:


 * 9. Transform: Heat inactivate the T4 DNA ligase in the ligated products by heating @ 65C for 15 minutes in a water bath. Transform the ligated products. Will be transforming 7 things instead of 8, because the base vector is in theory ligated or attached to a particular part in the ligation step. Will transform into TOP 10 Cells the following: (1) BV:TetR/p22 1, (2) BV:TetR/p22 2, (3) BV:Lac/LAMBDA, (4) BV:LacI 1, (5) BV:LacI 2, (6) BV:TetR 1, (7) BV:TetR 2. The following steps will take place for transforming 1-7:
 * a. Thaw TOP10 cells and incubate transformant (1-7) DNA on ice
 * b. Combine 50uL thawed cells with 50uL DNA and mix gently.
 * c. Incubate samples on ice for 20 minutes.
 * d. Heat shock cells in a 42C water bath for 1 minute.
 * e. Incubate samples on ice for 5 minutes.
 * f. Recover cells in 1.0mL 2xTy media, place cultures in incubator @ 37C for 1 hour with shaking @ 220rpm.
 * 10. Plate transformed TOP10 Cultures: Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.
 * d. Heat shock cells in a 42C water bath for 1 minute.
 * e. Incubate samples on ice for 5 minutes.
 * f. Recover cells in 1.0mL 2xTy media, place cultures in incubator @ 37C for 1 hour with shaking @ 220rpm.
 * 10. Plate transformed TOP10 Cultures: Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.
 * f. Recover cells in 1.0mL 2xTy media, place cultures in incubator @ 37C for 1 hour with shaking @ 220rpm.
 * 10. Plate transformed TOP10 Cultures: Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.
 * 10. Plate transformed TOP10 Cultures: Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.
 * 10. Plate transformed TOP10 Cultures: Plate approximately 150uL of culture on kanamycin resistant plates O/N @ 37C.