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Team:Newcastle/Labwork/24 September 2009
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[[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] | [[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] | ||
=Formal Lab Session - 24th September 2009= | =Formal Lab Session - 24th September 2009= | ||
| - | ==<u>Stochastic | + | [[Image:Team Newcastle 2009 iGEM 24-09-09 IMG 1379.JPG|350px|center]] |
| + | <br> | ||
| + | =<font color="Orange"><u>Overview</u></font>= | ||
| + | <font color="Orange"> | ||
| + | *[[#Metal Sensor Team|Metal Sensor Team]] '''- ''' | ||
| + | <br> | ||
| + | *[[#Stochastic Switch Team|Stochastic Switch Team]] '''- ''' | ||
| + | <br> | ||
| + | *[[#Sporulation Tuning/Chassis Team|Sporulation Tuning/Chassis Team]] '''- ''' | ||
| + | <br> | ||
| + | </font> | ||
| + | <br> | ||
| + | ==<u>Metal Sensor Team</u>== | ||
| + | ===To Do list=== | ||
| + | # PCR clean up of ''cotC'' PCR done on the 23/09/09 | ||
| + | # Digest PCR clean up and also digest ''pMUTIN4'' with ''BamHI'' and ''HindIII'' | ||
| + | # PCR clean up both of these products | ||
| + | # Run all of both sample on gel and excise bands | ||
| + | # Extract bands from gel using GenElute's Gel Extraction kit | ||
| + | # Set up overnight ligations of ''pMUTIN4'' and ''cotC-GFP-smtA'' BioBrick | ||
| + | |||
| + | ==<u>Stochastic Switch Team</u>== | ||
Today we did minipreps of the ara/sspb/pSB1AT3 cultures which were then digested with EcoRI and SpeI. These were run on an 0.8% gel. We expected to see a ~600bp band as well as the plasmid backbone and the gel photograph seemed fairly convincing: | Today we did minipreps of the ara/sspb/pSB1AT3 cultures which were then digested with EcoRI and SpeI. These were run on an 0.8% gel. We expected to see a ~600bp band as well as the plasmid backbone and the gel photograph seemed fairly convincing: | ||
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[[Image:Team Newcastle240909 trans mini.png]] | [[Image:Team Newcastle240909 trans mini.png]] | ||
| - | We set up a 50ml midiprep culture from the culture corresponding to lane 9 which seemed to have the clearest band. | + | We set up a 50ml midiprep culture from the culture corresponding to lane 9 which seemed to have the clearest band. |
| - | ==<u> Chassis | + | ==<u>Sporulation Tuning/Chassis Team</u>== |
===Introduction=== | ===Introduction=== | ||
| + | [[Image:Team Newcastle 2009 iGEM 24-09-09 IMG 1373.JPG|200px|right]] | ||
* We prepared kinA and pGFP-rrnB digested fragments yesterday, we'll carry on the next step as gel extraction and ligation and transformation. | * We prepared kinA and pGFP-rrnB digested fragments yesterday, we'll carry on the next step as gel extraction and ligation and transformation. | ||
* For the double clone part, we need to ligate it to pMutin4 vector. | * For the double clone part, we need to ligate it to pMutin4 vector. | ||
| Line 28: | Line 50: | ||
* Ligate digested PCR clean-up psc fragment to pMutin4 | * Ligate digested PCR clean-up psc fragment to pMutin4 | ||
** this psc fragment refer to the cwlJ:sleB fragment with HindIII and BamHI restriction sites at the end of each side. | ** this psc fragment refer to the cwlJ:sleB fragment with HindIII and BamHI restriction sites at the end of each side. | ||
| + | |||
| + | T4 ligase Buffer 2ul | ||
| + | Vector 2.5ul | ||
| + | insert DNA 14.5ul | ||
| + | T4 ligase 1ul | ||
| + | --------------------------- | ||
| + | 20ul | ||
| + | * -4C frige overnight. | ||
==== Prepare the culture for Mini Prep ==== | ==== Prepare the culture for Mini Prep ==== | ||
Latest revision as of 03:51, 22 October 2009
Project
Sub-projects
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Meetings
Planning
Links
Formal Lab Session - 24th September 2009
Overview
Metal Sensor Team
To Do list
- PCR clean up of cotC PCR done on the 23/09/09
- Digest PCR clean up and also digest pMUTIN4 with BamHI and HindIII
- PCR clean up both of these products
- Run all of both sample on gel and excise bands
- Extract bands from gel using GenElute's Gel Extraction kit
- Set up overnight ligations of pMUTIN4 and cotC-GFP-smtA BioBrick
Stochastic Switch Team
Today we did minipreps of the ara/sspb/pSB1AT3 cultures which were then digested with EcoRI and SpeI. These were run on an 0.8% gel. We expected to see a ~600bp band as well as the plasmid backbone and the gel photograph seemed fairly convincing:
We set up a 50ml midiprep culture from the culture corresponding to lane 9 which seemed to have the clearest band.
Sporulation Tuning/Chassis Team
Introduction
- We prepared kinA and pGFP-rrnB digested fragments yesterday, we'll carry on the next step as gel extraction and ligation and transformation.
- For the double clone part, we need to ligate it to pMutin4 vector.
Experiment procedure
Gel extraction
- Follow the standard procedure of Gel Extraction Kit(Sigma).
Ligation
- Ligate kinA to pGFP-rrnB
- Ligate digested PCR clean-up psc fragment to pMutin4
- this psc fragment refer to the cwlJ:sleB fragment with HindIII and BamHI restriction sites at the end of each side.
T4 ligase Buffer 2ul
Vector 2.5ul
insert DNA 14.5ul
T4 ligase 1ul
---------------------------
20ul
- -4C frige overnight.
Prepare the culture for Mini Prep
- Since we got colonies from yesterday's transformation, we need to culture the colonies for mini prep.
Prepare smm medium
- The protocal of making smm medium is come from our lab's B.subtilis 168 transformation protocal.
Conclusion
- After the autoclave of smm medium, the medium got cloudy. It may has something wrong with the ingredients.
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News
Events
- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
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- Newcastle iGEM Twitter
- [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
- [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]
