IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/20: Difference between revisions

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{|{{table}} width="800"
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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>}}
|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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==Entry title==
==Check Lux components==
* Insert your content here.


- Single colony PCR for :


*R0040 (MIT stuff)
*R0062 (MIT stuff)
*4 different colonies of S0168 (from a transformation plate from 12/08)
*4 different colonies of J04630 (from a transformation plate from 12/08)
- Protocol : add 1μL of cells (diluted in water), 10μL of Master Mis, 7μL of SDW, 1μL of VF primer and 1μL of VR primer
- Gel PCR products
Gel 1
* Lane2 : Hyperladder1
* Lane3 : JO4630, colony 1
* Lane4 : JO4630, colony 2
* Lane5 : JO4630, colony 3
* Lane6 : JO4630, colony 4
* Lane7 : HyperladderI
[[Image:photon.gif|300px|center]]
Gel 2
* Lane2 : HyperladderI
* Lane3 : ECE190 double digest
* Lane4 : S0168, colony 1
* Lane5 : S0168, colony 2
* Lane6 : S0168, colony 3
* Lane7 : S0168, colony 4
* Lane8 : HyperladderI
* Lane9 : R0040
* Lane10 : R0062
* Lane11 : Ladder 100bp
[[Image:photoo.gif|300px|center]]
- '''Results'''
* R0040 and R0062 : one big band of about 300b (expected size 293), OK!
* S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168
* J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!
* J04630 (colony 1) : one good band plus another band...
* J04630 (colony 3) : one band of about 600b, bad!
==Ligation==
- Materials :
*AgrA
*AgrB
*AgrC
*AgrD
*Pupp
*Pspac
*Ppac
*Pxyl
*RBS S
*RBS W
*psB4C5
- Double digest of PCR products
- Run vector, AgrA and AgrD on a gel
- DNA clean and concentrator for AgrA, B,C and D, promoters
- Microclean for both RBS
- Nanodrop
{|class="wikitable" style="text-align:center" border="1"
|-
! !! 260/280 !! ng/μL
|-
| AgrA || 1.66 || 16.4
|-
| AgrB || 1.91 || 23.5
|-
| AgrC || 1.99 || 35.9
|-
| AgrD || 2.13 || 4.9
|-
| Pxyl || 1.54 || 5.6
|-
| Ppac || 1.49 || 4.6
|-
| Pspc || 1.62 || 9.6
|-
| Pupp || 1.88 || 8.5
|-
| RBS S || 2.44 || 29
|-
| RBS W || 1.44 || 10.7
|-
|}
- Extract plasmid annd Agr from gel and clean
- Ligation


<!-- ## Do not edit below this line unless you know what you are doing. ## -->
<!-- ## Do not edit below this line unless you know what you are doing. ## -->

Revision as of 08:54, 5 September 2008

 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Check Lux components

- Single colony PCR for :

  • R0040 (MIT stuff)
  • R0062 (MIT stuff)
  • 4 different colonies of S0168 (from a transformation plate from 12/08)
  • 4 different colonies of J04630 (from a transformation plate from 12/08)

- Protocol : add 1μL of cells (diluted in water), 10μL of Master Mis, 7μL of SDW, 1μL of VF primer and 1μL of VR primer

- Gel PCR products

Gel 1

  • Lane2 : Hyperladder1
  • Lane3 : JO4630, colony 1
  • Lane4 : JO4630, colony 2
  • Lane5 : JO4630, colony 3
  • Lane6 : JO4630, colony 4
  • Lane7 : HyperladderI

Gel 2

  • Lane2 : HyperladderI
  • Lane3 : ECE190 double digest
  • Lane4 : S0168, colony 1
  • Lane5 : S0168, colony 2
  • Lane6 : S0168, colony 3
  • Lane7 : S0168, colony 4
  • Lane8 : HyperladderI
  • Lane9 : R0040
  • Lane10 : R0062
  • Lane11 : Ladder 100bp
File:Photoo.gif

- Results

  • R0040 and R0062 : one big band of about 300b (expected size 293), OK!
  • S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168
  • J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!
  • J04630 (colony 1) : one good band plus another band...
  • J04630 (colony 3) : one band of about 600b, bad!


Ligation

- Materials :

  • AgrA
  • AgrB
  • AgrC
  • AgrD
  • Pupp
  • Pspac
  • Ppac
  • Pxyl
  • RBS S
  • RBS W
  • psB4C5

- Double digest of PCR products

- Run vector, AgrA and AgrD on a gel

- DNA clean and concentrator for AgrA, B,C and D, promoters

- Microclean for both RBS

- Nanodrop

260/280 ng/μL
AgrA 1.66 16.4
AgrB 1.91 23.5
AgrC 1.99 35.9
AgrD 2.13 4.9
Pxyl 1.54 5.6
Ppac 1.49 4.6
Pspc 1.62 9.6
Pupp 1.88 8.5
RBS S 2.44 29
RBS W 1.44 10.7

- Extract plasmid annd Agr from gel and clean

- Ligation


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