PERFECTLY BLUNT CLONING OF DNA |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. Luria-Bertani (LB) media (1 L): | Mix 10 g of Bacto-tryptone, 5 of Yeast extract, and 10 g of NaCl (for taste). pH to 7.5 w/ NaOH. And dH2O to 1 L (Autoclave) |
| 2. LB-agar plates (500 mL): | After autoclaving LB media, add: 1.75 mL of 20 mg/mL X-gal (70 mg/mL), 400 mL of 100mM IPTG (80mM), and 600 mL of 12.5 mg/mL Tet (15 mg/mL) (For color selection). For vectors pT7B-3 (Kan) or pT7B2 (Amp), add the following selection: 300 mL of 50mg/mL Kan (30 mg/mL) or 250 mL of 100 mg/mL Amp (50 mg/mL). Pour plates |
| PROCEDURE | |
| This kit uses a vector prepared commercially that has been cut open and dephosphorylated so that any DNA fragment can be cloned into it. From Novagen | |
| 1. End conversion reaction: add 2 mL of PCR reaction, 5 mL of end conversion mix and dH2O to 10 mL | |
| 2. Incubate reaction at RT for 5 mins | |
3. Stop reaction by incubating at 65oC for 5 mins. Cool on ice for 2 mis |
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| 4. Ligate DNA to vector: transfer reaction to a new tube and add 1 mL of pT7Blue vector and 1 mL of Ligase | |
| 5. Incubate at RT for 2 hours | |
| 6. Add 1mL of ligation reaction to competent cells, place tubes on ice for 5 mins, heat shock at 42oC for 30 secs, place on ice for 2 mins, mix cells with 200mL of SOC media and shake at 37oC for 30 mins | |
| 7. Plate transformed cells on plates containing (blue/white screening) | |
8. Incubate plates at 37oC for 15 hours (inverted) and then place at 4oC for color development |
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| 9. Pick white colonies and analyze | |