Silver: Basic UNS Protocol: Difference between revisions
(New page: '''Basic UNS-Guided Assembly Protocol''' This very basic protocol will be useful to those experienced in restriction cloning methods, and focuses on the use of standardized part and desti...) |
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This very basic protocol will be useful to those experienced in restriction cloning methods, and focuses on the use of standardized part and destination vectors to accomplish UNS-guided assembly. A more detailed protocol will be presented in a forthcoming publication. | This very basic protocol will be useful to those experienced in restriction cloning methods, and focuses on the use of standardized part and destination vectors to accomplish UNS-guided assembly. A more detailed protocol will be presented in a forthcoming publication. | ||
1. Select an appropriate | 1. Select an appropriate [http://openwetware.org/wiki/Available_Vectors Destination Vector] for your application (e.g. pDesET for a medium-copy vector in E. coli, pDestRmceBAC for genomic integration into mammalian cells). | ||
2. Select an appropriate set of [http://openwetware.org/wiki/Available_Vectors Part Vector] (Use pJT series for pDestET or pDestBAC destination vectors, or pFL series for pDestRmceBAC or pDestPBBAC destination vectors. Mixing and matching may cause mismatches during assembly that decrease assembly efficiency). | |||
3. Clone sequences of interest (e.g. promoters/RBSes/genes/terminators) into part vectors as desired. Part vectors have a mix of BioBrick, BglBrick and other restriction sites to facilitate cloning. | |||
4. Digest cloned part vectors to generate linear, UNS-flanked parts. Digest destination vector as well. See Digestion Guide for details. | |||
5. Gel purify linear parts. Gel or PCR purify destination vector (we recommend the latter). | |||
6. Mix between 5 and 20 ng/kb of each purified part and destination vector in a total volume of 5 uL. | |||
7. Mix 5 uL 2x Isothermal Assembly Mixture with the 5 uL of DNA and pipette well to mix. Leave on a thermal cycler at 50 degrees Celsius with a hot-lid set to 105 Celsius for 1 h. | |||
8. Put the reaction on ice. Transform 1 uL of it into commercially available recA- cells (e.g. Mach1 or TOP10 chemically competent cells) or 5 uL into homemade competent cells (e.g. TSS Competent Cells). | |||
Revision as of 12:22, 22 April 2014
Basic UNS-Guided Assembly Protocol
This very basic protocol will be useful to those experienced in restriction cloning methods, and focuses on the use of standardized part and destination vectors to accomplish UNS-guided assembly. A more detailed protocol will be presented in a forthcoming publication.
1. Select an appropriate Destination Vector for your application (e.g. pDesET for a medium-copy vector in E. coli, pDestRmceBAC for genomic integration into mammalian cells).
2. Select an appropriate set of Part Vector (Use pJT series for pDestET or pDestBAC destination vectors, or pFL series for pDestRmceBAC or pDestPBBAC destination vectors. Mixing and matching may cause mismatches during assembly that decrease assembly efficiency).
3. Clone sequences of interest (e.g. promoters/RBSes/genes/terminators) into part vectors as desired. Part vectors have a mix of BioBrick, BglBrick and other restriction sites to facilitate cloning.
4. Digest cloned part vectors to generate linear, UNS-flanked parts. Digest destination vector as well. See Digestion Guide for details.
5. Gel purify linear parts. Gel or PCR purify destination vector (we recommend the latter).
6. Mix between 5 and 20 ng/kb of each purified part and destination vector in a total volume of 5 uL.
7. Mix 5 uL 2x Isothermal Assembly Mixture with the 5 uL of DNA and pipette well to mix. Leave on a thermal cycler at 50 degrees Celsius with a hot-lid set to 105 Celsius for 1 h.
8. Put the reaction on ice. Transform 1 uL of it into commercially available recA- cells (e.g. Mach1 or TOP10 chemically competent cells) or 5 uL into homemade competent cells (e.g. TSS Competent Cells).
