IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/13: Difference between revisions

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|style="background-color: #EEE"|[[Image:IgemCam.jpg|200px]]<span style="font-size:22px;"> Turing Pattern Formation</span>
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==Results for Starch plates==


- Add 5mL of iodine


- 1A1 or IA751 : big zones of clearance


- IA751 + ECE112 : no zones of clearance (photos), just small white points on colonies : the gene AmyE seems to be knocked out, transformation ok!
==PCR Agr A, B, C, D==


- 1A1 + ECE112 : problem, soft agar melted... impossible to observe!


 
Doing a PCR directly from the old Biobricks has worked for all parts of the operon. No prior digestion seems to be needed.
==Transformation==
 
- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171
 
- Spectrophotometer : blank made with medium A


{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
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! Time (min) !! 0 !! 20 !! 40 !! 60 !! 80 !! 100 !! 120
! Part !! 260/280 !! μg/mL
|-
|-
| OD650 || 0.1487 || 0.1541 || 0.1642 || 0.1980 || 0.2470 || 0.3205 || 0.3643
| A || 1.78 || 92.5
|-
|-
|}
| B || 1.87|| 96.5
 
- t0 = 120min
 
- Follow the protocol of transformation
 
- Plasmid miniprep of ECE153, 166, 171 (to make the transformation)
 
- Nanodrop ( to add 0.5μg of DNA for transformation)
 
{|class="wikitable" style="text-align:center" border="1"
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!  Vetor !! 260/280 !! μg/mL !! quantity of DNA to add for transformation (μL)
| C || 1.9 || 127.7
|-
|-
| ECE153 || 1.5 || 18.4 || 27.2
| D || 1.82 || 26.3
|-
| ECE166 || 1.83 || 39.5 || 12.7
|-
| ECE171 || 1.83 || 115.8 || 4.4
|}
 
- Plate (with appropriate antibiotics)
* ECE166 : Cm5
* ECE153 : Spc50
* ECE171 : Kan5 (to prepare agar plate, 40μL of Kan 25mg/mL into 200mL of agar)
 
==Check our stock of ECE171==
 
- We made big stocks of ECE171, before keeping it, we wanted to check it, so we run double digest on a gel!!
 
- Results : problem!!
 
- 2 possible causes : Double digest was made " days before and kept in the freezer (possible degradation) + too much DNA on the gel
 
- New double digest (from pellets in the freezer) + new gel
 
- Results : ok!
 
==Glycerol stocks==
 
- for IA751 and IA771, add 100μL of culture and 500μL of glycerol (60%)
 
==Xylose experiment==
 
- To test the transformation of ECE153, we want to induce the promoter Pxyl, and we will have green fluorescence
 
- Add 1mL of culture IA751 + ECE153 (from yesterday), 8mL of LB and 45μL of Spc50, 1mL of xylose (1g into 10mL)
 
==Plasmid miniprep for B.S.==
 
- We first tried to use the same protocol than for E.coli (Zyppy kit) for ECE166 transformed in IA751
 
- Nanodrop to see the result
 
{|class="wikitable" style="text-align:center" border="1"
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!  Vetor !! 260/280 !! μg/mL
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| ECE166, colony 1 || 1.69 || 11.2
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| || 1.41|| 14.5
|-
| ECE166, colony 2|| 1.34 || 7.0
|}
 
- We have very low concentration of DNA, we will digest that plasmid tomorrow morning to see if it is ECE166, and if it does work, we will try to modify our protocole tomorrow
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Revision as of 08:37, 5 September 2008

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PCR Agr A, B, C, D

Doing a PCR directly from the old Biobricks has worked for all parts of the operon. No prior digestion seems to be needed.

Part 260/280 μg/mL
A 1.78 92.5
B 1.87 96.5
C 1.9 127.7
D 1.82 26.3


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