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Richard Lab:Amplified insert assembly: Difference between revisions
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Richard Lab:Amplified insert assembly (view source)
Revision as of 21:26, 18 March 2011
, 18 March 2011→Procedure
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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).<br> | 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).<br> | ||
2. PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling).<br> | 2. PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling).<br> | ||
::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).<br> | ::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).<br> | ||
::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).<br> | ::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).<br> | ||
::*Only run 25-30 cycles as this will help ensure high fidelity.<br> | ::*Only run 25-30 cycles as this will help ensure high fidelity.<br> | ||
3. [[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours.<br> | 3. [[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours.<br> | ||
4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]].<br> | 4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]].<br> | ||
5. [[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br> | 5. [[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br> | ||
6. Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.<br> | 6. Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.<br> | ||
7. Kill all reactions by incubating for 20 mins at 80°C.<br> | 7. Kill all reactions by incubating for 20 mins at 80°C.<br> | ||
8. [[Richard Lab:Ligation|Ligate]] at a molar ratio of 4:1 (insert:vector).<br> | 8. [[Richard Lab:Ligation|Ligate]] at a molar ratio of 4:1 (insert:vector).<br> | ||
9. [[Richard Lab:Electroporation of E. coli|Transform]].<br> | 9. [[Richard Lab:Electroporation of E. coli|Transform]].<br> | ||
10. Plate on plates with the same antibiotic as the "vector" resistance.<br> | 10. Plate on plates with the same antibiotic as the "vector" resistance.<br> | ||
11. Celebrate. | 11. Celebrate. | ||
