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Minnesota/24 July 2008
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| - | !| Parts !! 10x Buffer !! H20 !! Base Vector !! Insert DNA !! T4 DNA Ligase !! Total Volume | + | !| Parts !! 10x Buffer !! H20 !! Base Vector !! Insert DNA !! T4 DNA Ligase !! Total Volume |
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| BV + TetR 1 || 4.0uL || 13.0uL || 5.0uL || 17.0uL || 1.0uL || 40.0uL | | BV + TetR 1 || 4.0uL || 13.0uL || 5.0uL || 17.0uL || 1.0uL || 40.0uL | ||
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| BV + TetR/p22 2 || 4.0uL || 6.0uL || 5.0uL || 24.0uL || 1.0uL || 40.0uL | | BV + TetR/p22 2 || 4.0uL || 6.0uL || 5.0uL || 24.0uL || 1.0uL || 40.0uL | ||
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| + | |'''9. Transform:''' Transform the ligated products. | ||
Revision as of 23:35, 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:
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| 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:
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| 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:
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| 9. Transform: Transform the ligated products. |
