IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/07/25: Difference between revisions

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<div id="about">
{{Cambridge08a}}
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__NOTOC__
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|style="background-color: #EEE"|[[Image:IgemCam.jpg|200px]]<span style="font-size:22px;"> Turing Pattern Formation</span>
|style="background-color: #444444"|[[Image:Signalling_button.gif|200px|center]]
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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* Antibiotic test


'''Results for Amp'''
'''Results'''


Even with a concentration of 10μg/mL, Amp kills Bacillus. Since we want to know which is the lowest concentration of Amp which kills B.S, we are going to test with some lower concentrations.
Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!


'''Results for Cm'''


Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm.


*Test if biobrick of last year are really the one we want


=Wet Work=
- Yesterday : Single colony of I746001 and I746101 transferred in 10mL of LB + Amp (final concentration 100μg/mL)
- Take 8mL of I746001 (AIP sender) and of I746101
- Centrifuge them
- Saved 2ml of each in tubes to regrow and add LB
- Miniprep I746001 and I746101 pellets (plasmid mniprep kit) : same protocol but 60μL of Zyppy Glution Buffer in step 8


* New antibiotic tests
- Restriction Digest : use EcoI and Spel restriction enzymes
- Add sequentially : 15μL of SDW + 2μL of 10X Fast Digest Buffer + 2μL of DNA + 1μL of EcoI + 1μL of Spel
- Microfuge tubes
- Incubate for 10min at 37°C
- Heat inactivation of enzymes at 80°C for 5min


'''Dilution of Amp'''
- GEL
{|class="wikitable" style="text-align:center" border="1"
20μLof samples + 4μL of dye
|-
! Concentration (μg/mL) !! 10 !! 7.5 !! 5 !! 2.5 !! 1
|-
| 10 μg/mL Stock (from yesterday) || 1:1 || 3:4 || 1:2 || 1:4 || 1:10
|-
|}




'''Dilution of Cm'''
* Grow 1A1
 
{|class="wikitable" style="text-align:center" border="1"
|-
! Concentration (μg/mL) !! 35 !! 37.5 !! 40 !! 42.5 !! 45
|-
| 35 μg/mL Stock (from yesterday) || 1:1 || 5:6 (from 45μg/mL) || 1:875 || 17:18 (from 45μg/mL) || 9:7000
|-
|}


- 10mL of LB + 15μL of 1A1
- Grow at 37°C


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Revision as of 04:35, 5 September 2008

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Results from Yesterday

  • Kan resistance of biobricks
Biobrick Antibiotic Contration (μg/mL) Observations Conclusion
I746001 Kan 25 Many colonies Kan Resistance OK
I746101 Kan 25 Many colonies Kan Resistance OK
Control PUC19 Kan 25 No colony No Kan Resistance


Results

Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!


  • Test if biobrick of last year are really the one we want

- Yesterday : Single colony of I746001 and I746101 transferred in 10mL of LB + Amp (final concentration 100μg/mL) - Take 8mL of I746001 (AIP sender) and of I746101 - Centrifuge them - Saved 2ml of each in tubes to regrow and add LB - Miniprep I746001 and I746101 pellets (plasmid mniprep kit) : same protocol but 60μL of Zyppy Glution Buffer in step 8

- Restriction Digest : use EcoI and Spel restriction enzymes - Add sequentially : 15μL of SDW + 2μL of 10X Fast Digest Buffer + 2μL of DNA + 1μL of EcoI + 1μL of Spel - Microfuge tubes - Incubate for 10min at 37°C - Heat inactivation of enzymes at 80°C for 5min

- GEL 20μLof samples + 4μL of dye


  • Grow 1A1

- 10mL of LB + 15μL of 1A1 - Grow at 37°C


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