Team:Cornell/Notebook/Nah operon

From 2012.igem.org

(Difference between revisions)
(June 10th-16th)
(June)
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===June 10th-16th===
===June 10th-16th===
====June 13th, Wednesday====
====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.
+
:Set up [http://2011.igem.org/Team:Cornell/Protocols 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 using Phusion polymerase, an alternate method of mutation using Phusion will be pursued.
:If Gibson Assembly fails, but we are able to PCR the entire 10kb nah operon using Phusion polymerase, an alternate method of mutation using Phusion will be pursued.
::''Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati''
::''Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati''
====June 14th, Thursday====
====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.
+
: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 [http://2011.igem.org/Team:Cornell/Protocols PCR] with shorter extension time.
====June 15th, Friday====
====June 15th, Friday====
Line 20: Line 20:
====June 19th, Tuesday====
====June 19th, Tuesday====
:Ran Gibson Assembly of nah operon fragments into pBMT-1 backbone and transformed into DH5a electrocompotent ''E. coli'' cells.  
: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.  
+
:Set up [http://2011.igem.org/Team:Cornell/Protocols 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 [http://2011.igem.org/Team:Cornell/Protocols digestion] of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.  
:We'll also check to see if we got colonies from the transformation then.
:We'll also check to see if we got colonies from the transformation then.
::''Work done by: Dylan and Swati''
::''Work done by: Dylan and Swati''
Line 30: Line 30:
===June 24th-30th===
===June 24th-30th===
====June 27th, Wednesday====
====June 27th, Wednesday====
-
*Ran digest of gibson-assembled nah operon on gel
+
*Ran digest of gibson-assembled nah operon with [http://2011.igem.org/Team:Cornell/Protocols gel electrophoresis]
====June 28th, Thursday====
====June 28th, Thursday====

Revision as of 20:46, 28 June 2012

Home Team Project Parts Modeling Notebook Protocols Safety Attributions

Contents

June

Project Description

The purpose of this subproject is to make the nah operon biobrick compatible for the use of future iGEM teams. The nah operon is an operon that converts napthalene into salicylate. In our system, this will allow napthalene to be detected by the salicylate reporter. There are three standard cutsites internal to the operon which we are removing - the biobrick compatible piece could be used by future teams in bioremediation projects.

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 using Phusion polymerase, an alternate method of mutation using Phusion 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.

June 17th-23rd

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.
We'll also check to see if we got colonies from the transformation then.
Work done by: Dylan and Swati

June 24th-30th

June 27th, Wednesday

====June 28th, Thursday====