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Team:Cornell/Notebook/Nah operon/June10
From 2012.igem.org
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=June 10th-16th= | =June 10th-16th= | ||
==June 13th, Wednesday== | ==June 13th, Wednesday== | ||
| - | *Set up [[Team:Cornell/Notebook/ | + | *Set up [[Team:Cornell/Notebook/Phusion_PCR|PCR]] for four fragments of the nah operon as well as the pBMT-1 backbone, using primers designed to mutate cut-sites concurrently with Gibson Assembly. Also set up a PCR for the entire nah operon. Due to length of the fragments, a longer extension time was chosen. |
*If Gibson Assembly fails, but we are able to PCR the entire 10kb nah operon, an alternate method of mutation using three sequential site-directed mutageneses will be pursued. | *If Gibson Assembly fails, but we are able to PCR the entire 10kb nah operon, an alternate method of mutation using three sequential site-directed mutageneses will be pursued. | ||
**''Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati'' | **''Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati'' | ||
Revision as of 21:56, 3 July 2012
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The purpose of this subproject is to make the nah operon biobrick compatible for the use of future iGEM teams. The nah operon codes for a pathway that converts naphthalene into salicylate. In our system, this will allow naphthalene to be detected by the salicylate reporter. There are three standard cut sites internal to the operon which we are removing - PstI, and two NotI sites. This biobrick compatible piece could be useful to future teams in projects that involve bioremediation or detection of naphthalene. Back to nah operon week view
June 10th-16th
June 13th, Wednesday
- Set up PCR for four fragments of the nah operon as well as the pBMT-1 backbone, using primers designed to mutate cut-sites concurrently with Gibson Assembly. Also set up a PCR for the entire nah operon. Due to length of the fragments, a longer extension time was chosen.
- If Gibson Assembly fails, but we are able to PCR the entire 10kb nah operon, an alternate method of mutation using three sequential site-directed mutageneses will be pursued.
- Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati
June 14th, Thursday
- PCR only amplified nah1, nah3, and nah4 - longer fragments (nah2, the full nah operon, and pBMT-1) were not identified on the PCR product gel. Set up another PCR with shorter extension time.
June 15th, Friday
- PCR of four out of five products visible on gel. Set up PCR of pBMT-1, final fragment required for Gibson Assembly of the nah operon.
