IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/29: Difference between revisions

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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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- run again on a e-gel
- run again on a e-gel


*Result : nothing, just the primers! Problem with our PCR
* Lane1 :ladder 100bp
* Lane2 : Pupp colony 1
* Lane3 : Pupp colony 3
* Lane4 : Pspac colony 1
* Lane5 : Pspac colony 3
* Lane6 : RBS S colony 1
* Lane7 : RBS S colony 4
* Lane8 : RBS W colony 1
* Lane9 : RBS W colony 4
* Lane10 : agrD colony 1
* Lane11 : agrD colony 4
* Lane12 : HyperladderI


==Stock of ECE112==
[[Image:photor.gif|300px|center]]


- Bulk up ECE112 (Amp100) rfom 10mL LB (28/08), take 1mL and add to 100mL of LB in big flask
*Result : nothing, just the primers! Problem with our PCR
 
- Grow overnight in 37°C incubator with vigorous shaking
 
- Aliquot in 10mL tubes, pellet and freeze cell pellets
 
==Transformation of glycerol stock==
 
* Strian : IA751
 
- TTransfer into Eppendorf tubes and spin down for 30min
 
-Remove glycerol and add medium B
 
- Add ECE112 and ECE153, and one control tube
 
- incubate for 30min in 37°C
 
- Plate out ECE112 (Cm5), ECE153 (Spc50), DNA less (blank, Cm5, Spc50)


==Plate biobricks from MIT==
==Plate biobricks from MIT==

Revision as of 09:09, 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>

Results of transformation with our ligation products

  • Everything grew! Better efficiency with electrop. than with chemical protocol

Single colony PCR to check our transformation

Transformed products number of picked colonies from chemical transformation number of picked colonies from electrop. transformation (neat) number of picked colonies from electrop. transformation (1:10)
Pupp 2 2 0
Pspac 2 2 0
RBS S 3 0 2
RBS W 3 0 2
agrD 3 2 0

- Single colony PCR : 13μL of SDW+cells, 5μL of Master Mix, 1μL of VF and 1μL of VR (and plate each single colony)

- Load a gel (1.3% agarose) : 5μL of PCR products + 1μL of dye (only 1μL of 100b ladder)

In the death plasmid, the VF-VR size is about 280b.

Transformed products size of the product (with cutting sites) expected size after PCR (about)
Pupp 255 480
Pspac 125 350
RBS S 56 280
RBS W 56 280
agrD 200 430
  • Result : nothing, even no ladder, problem with the gel!

- run again on a e-gel

  • Lane1 :ladder 100bp
  • Lane2 : Pupp colony 1
  • Lane3 : Pupp colony 3
  • Lane4 : Pspac colony 1
  • Lane5 : Pspac colony 3
  • Lane6 : RBS S colony 1
  • Lane7 : RBS S colony 4
  • Lane8 : RBS W colony 1
  • Lane9 : RBS W colony 4
  • Lane10 : agrD colony 1
  • Lane11 : agrD colony 4
  • Lane12 : HyperladderI
  • Result : nothing, just the primers! Problem with our PCR

Plate biobricks from MIT

  • E0840
  • B0014
  • I712007
  • C0012
  • B0015
  • C0061
  • R0063


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