IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/13

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(In Preparation of Beta-galactosidase Assay)
Line 114: Line 114:
Biobrick Extraction:
Biobrick Extraction:
-
*  
+
E0040
 +
* GFP (Amp resistance)
 +
* Extracted from 2007 Plate 1 Well 5H
 +
I13522
 +
* E.coli constitutive promoter with RBS and GFP (Amp resistance)
 +
* Extracted from 2007 Plate 3 Well 13C
 +
B0034
 +
* E. coli RBS (Amp resistance)
 +
* Extracted from 2008 Plate 1000 Well 2E
 +
R0040
 +
* E. coli promoter (Amp resistance)
 +
* Extracted from 2008 Plate 1000 Well 4C
 +
Transformation of competent TOP10 with the 4 biobricks above along with pUC19 control
 +
* 20μL TOP10 used for each transformation with 2μL DNA
 +
* Followed standard protocol
 +
* Plated out neat and 1/10 after 2 hours of incubation, then plates are kept at 37°C overnight
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Revision as of 13:19, 8 September 2008



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Contents

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!

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


Transformation of IA751

- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171

- Spectrophotometer : blank made with medium A

Time (min) 0 20 40 60 80 100 120
OD650 0.1487 0.1541 0.1642 0.1980 0.2470 0.3205 0.3643

- 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)

Vetor 260/280 μg/mL quantity of DNA to add for transformation (μL)
ECE153 1.5 18.4 27.2
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

Vetor 260/280 μg/mL
ECE166, colony 1 1.69 11.2
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

Transformation of ECE188, 189, 190 in E.coli

  • We received this vector in DNA stocks, so we have to transform them to test them 9because we do not have enough DNA)
  • Use TOP10 competent cells
  • Pellet, add 100μL of CaCl2 solution, for each transformation, use 50μL of cells
  • Add 2μL of DNA, and 1μL of PUC19 for control
  • 30min on ice
  • 2min at 42°C, 2min on ice
  • 2h at 37°C
  • Plate on Amp100 plates

In Preparation of Beta-galactosidase Assay

Biobrick Extraction: E0040

  • GFP (Amp resistance)
  • Extracted from 2007 Plate 1 Well 5H

I13522

  • E.coli constitutive promoter with RBS and GFP (Amp resistance)
  • Extracted from 2007 Plate 3 Well 13C

B0034

  • E. coli RBS (Amp resistance)
  • Extracted from 2008 Plate 1000 Well 2E

R0040

  • E. coli promoter (Amp resistance)
  • Extracted from 2008 Plate 1000 Well 4C

Transformation of competent TOP10 with the 4 biobricks above along with pUC19 control

  • 20μL TOP10 used for each transformation with 2μL DNA
  • Followed standard protocol
  • Plated out neat and 1/10 after 2 hours of incubation, then plates are kept at 37°C overnight
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