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Team:Newcastle/Labwork/22 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 - 22nd September 2009= | =Formal Lab Session - 22nd September 2009= | ||
| - | ==<u>Stochastic | + | [[Image:Team Newcastle 2009 iGEM 22-09-09 IMG 1321.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>== | ||
| + | We carried out the following tasks: | ||
| + | <br> | ||
| + | * Digested ''cotC-GFP-smtA'' PCR products with ''BamHI'' and ''HindIII'' (FastDigest) and allowed them to incubate for 1 hour under 37C. | ||
| + | <br> | ||
| + | * Conducted PCR clean-up using GenElutes PCR clean-up kit | ||
| + | <br> | ||
| + | * Digested ''cotC-GFP-smtA'' with ''BamHI'' and ''HindIII'' | ||
| + | <br> | ||
| + | * Carried out FastLigations with the following concentrations: | ||
| + | # 2ul of ligase buffer | ||
| + | # 2.5ul of vector DNA (i.e. ''pMUTIN4'') | ||
| + | # 14.5ul of insert DNA (i.e. ''cotC-GFP-smtA'') | ||
| + | # 1ul of ligase | ||
| + | '''Please note there were three reactions: 1) vector + insert + ligase, 2) vector + ligase and 3) vector + NO ligase. | ||
| + | <br> | ||
| + | * We also started to make some competent ''E. coli'' cells by carrying out the 'Culture' step in [https://2009.igem.org/Team:Newcastle/Project/Labwork/PhilsProtocols#Preparing_Competent_E.coli_cells_for_Heat_Shock Phil Aldridge's preparing competent cells protocol] | ||
| + | <br> | ||
| + | ==<u>Stochastic Switch Team</u>== | ||
| + | [[Image:Team Newcastle 2009 iGEM 22-09-09 IMG 1324.JPG|200px|right]] | ||
Today we transformed'' E.coli'' JM109 cels with the overnight ligation product from yesterday using the promega protocol. We used the following controls: | Today we transformed'' E.coli'' JM109 cels with the overnight ligation product from yesterday using the promega protocol. We used the following controls: | ||
| Line 16: | Line 46: | ||
Today again we redid the digests of the sac miniprep however again there was no DNA present. | Today again we redid the digests of the sac miniprep however again there was no DNA present. | ||
| + | |||
| + | ==<u>Sporulation Tuning/Chassis Team</u>== | ||
| + | [[Image:Team Newcastle 2009 iGEM 22-09-09 IMG 1306.JPG|200px|right]] | ||
| + | ===Introduction=== | ||
| + | * After overnight culture of cells for Midi Prep, today we need Midi Prep L1 and L2 plasmid and digest the plasmid to check the Midi prep result. | ||
| + | <br> | ||
| + | |||
| + | ===Experiment procedure=== | ||
| + | |||
| + | ====Midi prep L1(pSB1AT3:cwlJ) and L2(pSB1AT3:cwlJ:sleB) ==== | ||
| + | * Before we carried out Midi Prep, we take out 1ml of cell mix and put them into 1.5ml eppendorf tube to keep in fridge. | ||
| + | * 50ml of cells were used for Midi Prep. | ||
| + | * Midi prep processed by following the standard protocal of Plasmid Midi Prep Kit(Sigma). | ||
| + | |||
| + | ====Digest Midi prep result ==== | ||
| + | * Using the same reaction volume as day 18/09/09 | ||
| + | |||
| + | ===Conclusion=== | ||
| + | * Both Midi prep for L1 and L1 are right plasmid. | ||
| + | * Nano dorp of our fragments and plasmid. | ||
| + | cwlJ:sleB fragment 53.5ng/ul | ||
| + | (after PCR using primer cwlJsleBForward and cwlJsleBReverse) | ||
| + | |||
| + | pMutin4 after digestion with HindIII and BamHI 24.9ng/ul | ||
| + | |||
| + | L1 Midi (plasmid pSB1AT3:cwlJ) 76.9ng/ul | ||
| + | |||
| + | L2 Midi (plasmid pSB1AT3:cwlJ:sleB) 55.0ng/ul | ||
| + | |||
| + | kinA fragment after digest(EcoRI and NheI) and gel extraction 6.1ng/ul | ||
| + | |||
| + | <br> | ||
| + | [[Image:Team_newcastle_2009_hanny_geldoc_220909_1.jpg |150px|center]] | ||
| + | |||
{{:Team:Newcastle/Project/Labwork/CalTemplate}} | {{:Team:Newcastle/Project/Labwork/CalTemplate}} | ||
Latest revision as of 03:42, 22 October 2009
Project
Sub-projects
Wet Lab
Meetings
Planning
Links
Formal Lab Session - 22nd September 2009
Overview
Metal Sensor Team
We carried out the following tasks:
- Digested cotC-GFP-smtA PCR products with BamHI and HindIII (FastDigest) and allowed them to incubate for 1 hour under 37C.
- Conducted PCR clean-up using GenElutes PCR clean-up kit
- Digested cotC-GFP-smtA with BamHI and HindIII
- Carried out FastLigations with the following concentrations:
- 2ul of ligase buffer
- 2.5ul of vector DNA (i.e. pMUTIN4)
- 14.5ul of insert DNA (i.e. cotC-GFP-smtA)
- 1ul of ligase
Please note there were three reactions: 1) vector + insert + ligase, 2) vector + ligase and 3) vector + NO ligase.
- We also started to make some competent E. coli cells by carrying out the 'Culture' step in Phil Aldridge's preparing competent cells protocol
Stochastic Switch Team
Today we transformed E.coli JM109 cels with the overnight ligation product from yesterday using the promega protocol. We used the following controls:
- Tube 1: Backbone + insert + ligase (proper ligation)
- Tube 2: Backbone + ligase (control for background ligation level)
- Tube 3: Backbone + insert (control for ligase effectiveness)
These were left in the incubator for an hour and then plated out (2 plates per tube 200ul and 50ul) onto Amp + Tet LB plated and put in the 37C incubator overnight.
Today again we redid the digests of the sac miniprep however again there was no DNA present.
Sporulation Tuning/Chassis Team
Introduction
- After overnight culture of cells for Midi Prep, today we need Midi Prep L1 and L2 plasmid and digest the plasmid to check the Midi prep result.
Experiment procedure
Midi prep L1(pSB1AT3:cwlJ) and L2(pSB1AT3:cwlJ:sleB)
- Before we carried out Midi Prep, we take out 1ml of cell mix and put them into 1.5ml eppendorf tube to keep in fridge.
- 50ml of cells were used for Midi Prep.
- Midi prep processed by following the standard protocal of Plasmid Midi Prep Kit(Sigma).
Digest Midi prep result
- Using the same reaction volume as day 18/09/09
Conclusion
- Both Midi prep for L1 and L1 are right plasmid.
- Nano dorp of our fragments and plasmid.
cwlJ:sleB fragment 53.5ng/ul (after PCR using primer cwlJsleBForward and cwlJsleBReverse) pMutin4 after digestion with HindIII and BamHI 24.9ng/ul
L1 Midi (plasmid pSB1AT3:cwlJ) 76.9ng/ul
L2 Midi (plasmid pSB1AT3:cwlJ:sleB) 55.0ng/ul
kinA fragment after digest(EcoRI and NheI) and gel extraction 6.1ng/ul
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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)
Social Net
- 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]
