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==Procedure== | ==Procedure== | ||
1. [[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using a [http://www.omegabiotek.com/products.php?CateID=11 kit] or [[Miniprep/GET buffer|this protocol]] (30 mins). | |||
2. PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling). | |||
::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent). | ::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent). | ||
::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C). | ::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C). | ||
::*Only run 25-30 cycles as this will help ensure high fidelity. | ::*Only run 25-30 cycles as this will help ensure high fidelity. | ||
3. [[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | |||
4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]]. | |||
5. [[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases). | |||
6. Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done. | |||
7. Kill all reactions by incubating for 20 mins at 80°C. | |||
8. [[Richard Lab:Ligation|Ligate]] at a molar ratio of 4:1 (insert:vector). | |||
9. [[Richard Lab:Electroporation of E. coli|Transform]]. | |||
10. Plate on plates with the same antibiotic as the "vector" resistance. | |||
11. Celebrate. | |||
*If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours. | *If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours. |
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