| 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:
| Parts | 10x Buffer | BSA | H20 | DNA from parts | RE 1 | RE 2 | [DNA] ng/uL
|
| BV | 5.0uL | 0.5uL | 30.8uL | 11.7uL | EcoRI, 1.0uL | Pst1, 1.0uL | 100.62 ng/uL
|
| TetR Pro. 1 | 5.0uL | 0.5uL | 35.5uL | 7.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 23.8 ng/uL
|
| Tet R Pro. 2 | 5.0uL | 0.5uL | 35.5uL | 7.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 23.8 ng/uL
|
| LacI Pro. 1 | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 168.0 ng/uL
|
| LacI Pro. 2 | 5.0uL | 0.5uL | 41.5uL | 1.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 168.0 ng/uL
|
| Lac/Lam 1 | 5.0uL | 0.5uL | 28.5uL | 14.0uL | EcoRI, 1.0uL | Pst1, 1.0uL | 9.8 ng/uL
|
| Tet/p22 1 | 5.0uL | 0.5uL | 34.0uL | 8.5uL | EcoRI, 1.0uL | Pst1, 1.0uL | 20.4 ng/uL
|
| Tet/p22 2 | 5.0uL | 0.5uL | 34.0uL | 8.5uL | EcoRI, 1.0uL | Pst1, 1.0uL | 20.4 ng/uL
|
|
| 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:
| Parts | Antarctic Phosphatase | Antarctic Phosphatase Buffer
|
| BV | 1.0uL | 5.1uL
|
|
| 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:
| Parts | 10x Buffer | H20 | Base Vector | Insert DNA | T4 DNA Ligase | Total Volume
|
| BV + TetR 1 | 4.0uL | 13.0uL | 5.0uL | 17.0uL | 1.0uL | 40.0uL
|
| BV + TetR 2 | 4.0uL | 6.0uL | 5.0uL | 24.0uL | 1.0uL | 40.0uL
|
| BV + LacI 1 | 4.0uL | 10.0uL | 5.0uL | 20.0uL | 1.0uL | 40.0uL
|
| BV + LacI 2 | 4.0uL | 15.0uL | 5.0uL | 15.0uL | 1.0uL | 40.0uL
|
| BV + Lac/LAMBDA | 4.0uL | 17.0uL | 5.0uL | 13.0uL | 1.0uL | 40.0uL
|
| BV + TetR/p22 1 | 4.0uL | 13.0uL | 5.0uL | 17.0uL | 1.0uL | 40.0uL
|
| BV + TetR/p22 2 | 4.0uL | 6.0uL | 5.0uL | 24.0uL | 1.0uL | 40.0uL
|
|
| 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.
|
"
