Team:Cornell/Notebook/Nah operon/June17

From 2012.igem.org

(Difference between revisions)
(June 17th-23rd)
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===June 17th-23rd===
===June 17th-23rd===
====June 17th, Sunday====
====June 17th, Sunday====
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*Successful PCR of pBMT-1 [[Gel Purification | gel purified]].
+
*Successful PCR of pBMT-1 [[Team:Cornell/Notebook/Gel_purification| gel purified]].
====June 19th, Tuesday====
====June 19th, Tuesday====
-
*Ran [[Gibson Assembly]] of nah operon fragments into pBMT-1 backbone and [[Transformation | transformed]] into DH5a electrocompotent ''E. coli'' cells.  
+
*Ran [[Team:Cornell/Notebook/Gibson_assembly|Gibson Assembly]] of nah operon fragments into pBMT-1 backbone and [[Team:Cornell/Notebook/Transformation_ecoli | transformed]] into DH5a electrocompotent ''E. coli'' cells.  
-
*Set up [[PCR]] to amplify the Gibson Assembly products.  
+
*Set up [[Team:Cornell/Notebook/PCR|PCR]] to amplify the Gibson Assembly products.  
**''Work done by: Dylan and Swati''
**''Work done by: Dylan and Swati''
====June 20th, Wednesday====
====June 20th, Wednesday====
-
*Set up a [[digestion]] of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
+
*Set up a [[Team:Cornell/Notebook/Digestion|digestion]] of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
*Three colonies on plates of DH5a transformed with Gibson Assembly product. Made [[liquid cultures]] to miniprep and sequence.
*Three colonies on plates of DH5a transformed with Gibson Assembly product. Made [[liquid cultures]] to miniprep and sequence.
**''Work done by: Dylan and Swati''
**''Work done by: Dylan and Swati''
====June 22nd, Friday====
====June 22nd, Friday====
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*[[Miniprep | Miniprepped]] directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
+
*[[Team:Cornell/Notebook/Miniprep | Miniprepped]] directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
-
*Ran undigested miniprep with [http://2011.igem.org/Team:Cornell/Protocols gel electrophoresis], looking for large bands corresponding to supercoiled DNA. The gel shows distinct bands for all three lanes. We interpreted this to mean that we got product. Submitted for [[sequencing]]
+
*Ran undigested miniprep with [[Team:Cornell/Notebook/Gel_electrophoresis|gel electrophoresis]], looking for large bands corresponding to supercoiled DNA. The gel shows distinct bands for all three lanes. We interpreted this to mean that we got product. Submitted for [[Team:Cornell/Notebook/Sequencing|sequencing]]
::[[File:Cornell2012_0621_Gibson-entire-plasmid.jpg|600px]]
::[[File:Cornell2012_0621_Gibson-entire-plasmid.jpg|600px]]

Revision as of 21:37, 3 July 2012

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Contents

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 17th-23rd

June 17th, Sunday

June 19th, Tuesday

  • Ran Gibson Assembly of nah operon fragments into pBMT-1 backbone and transformed into DH5a electrocompotent E. coli cells.
  • Set up PCR to amplify the Gibson Assembly products.
    • Work done by: Dylan and Swati

June 20th, Wednesday

  • Set up a digestion of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
  • Three colonies on plates of DH5a transformed with Gibson Assembly product. Made liquid cultures to miniprep and sequence.
    • Work done by: Dylan and Swati

June 22nd, Friday

  • Miniprepped directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
  • Ran undigested miniprep with gel electrophoresis, looking for large bands corresponding to supercoiled DNA. The gel shows distinct bands for all three lanes. We interpreted this to mean that we got product. Submitted for sequencing
Cornell2012 0621 Gibson-entire-plasmid.jpg