Team:Evry/Notebook/w3

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<h2>Tuesday, 26th June</h2>
<h2>Tuesday, 26th June</h2>
 +
 +
<h3>Bacterial Transformation:</h3>
 +
Petri dish
 +
 +
<ul>
 +
<li>YFP
 +
<li>GFP
 +
<li>CFP
 +
<li>RFP
 +
<li>pCS2 (+) from 22/06/12
 +
</ul>
<h2>Wednesday, 27th June</h2>
<h2>Wednesday, 27th June</h2>
 +
 +
<ol>
 +
<li>MiniPrep- followed by nanodrop: confirmation of DNA presence in transformed bacteria
 +
<li>Agarose gel electrophoresis (samples of 4 fluorescent DNA)
 +
<li>Inoculation of colonies in LB medium- incubation at 37 degrees overnight
 +
</ol>
<h2>Thursday, 28th June</h2>
<h2>Thursday, 28th June</h2>
 +
 +
<h3>Gel migration 1</h3>
 +
 +
<ul>
 +
<li>Gel preparation: 25mL agarose+TAE (freezer) + 2microL BET
 +
<li>Sample preparation:
 +
<ul>
 +
<li>DNA Ladder 1kB: 1microL ladder + 1microL loading buffer + 4microL ddH2O
 +
<li>Digested RFP, YFP, CFP, GFP, pCS2+ (from 28 june): 10microL sample + 2microL loading buffer
 +
</ul>
 +
<li>100V, 1h
 +
</ul>
 +
 +
<strong>Result:</strong> no DNA detectable, Solution: to redo the step of digestion with more DNA
 +
 +
<h3>DNA digestion:</h3>
 +
<ul>
 +
<li>4ul of CFP, GFP, RFP, YFP dosed on 28 june
 +
<li>3ul of pSC2+ dosed on 28 june
 +
</ul>
 +
 +
<h2>Gel migration 2:</h2>
 +
 +
<ul>
 +
<li>Gel preparation: 50mL agarose+TAE (freezer) + 4ul BET
 +
<li>Sample preparation:
 +
<ul>
 +
<li>DNA Ladder 1kB: 1ul ladder + 1ul loading buffer + 4ul ddH2O
 +
<li>Digested RFP, YFP, CFP, GFP, pCS2+ (from 29 june) : 15ul sample + 3ul loading buffer
 +
</ul>
 +
<li>100V, 1h
 +
</ul>
 +
 +
<strong>Result:<strong> no RFP detectable (see picture below):
 +
 +
<img src="https://static.igem.org/mediawiki/2012/8/8d/Photo_GelMigration2906.jpeg" alt:"migration results">
<h2>Friday, 29th June</h2>
<h2>Friday, 29th June</h2>

Revision as of 20:31, 2 August 2012

Weeks

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Week 3: 25th June - 1st July

Monday, 25th June

Bacterial Transformation

Bacteria:T10
Reporters:
  • RFP: P1-18F K
  • GFP: P1-14K A
  • CFP: P1-6A A
  • YFP: P2/24E AK
  • OFP: P2/13N K

  • Violecein P3/12B T
  • Vio operon ABDE P3/20H K
  • Vio operon ABCE P3/20J K
  • CelEBI w/RRS P3/6H A
  • CelEBY w/RRS P3/6L H
Protocol:
  1. Keep constantly the cells on ice
  2. Rehydratation of DNA in 10uL H2O 2) Add 1 uL of DNA in 50 uL of T10
  3. Incubate 30 min on ice
  4. Heat shock the cells during 30 sec at 42 degree Celsius in a water-bath without shaking
  5. Put 2 min on ice
  6. Add 500 uL of pre warmed SOC-medium
  7. Incubate 1h at 37 degree Celsius at 225 rpm
  8. Spin at 5000 rpm during 30 sec
  9. Remove 150 uL - 400 uL of supernatant
  10. Resuspend the pellet in the 150 uL left
  11. Spread on appropriate plates
  12. Incubate overnight at 37 degree Celsius

Tuesday, 26th June

Bacterial Transformation:

Petri dish
  • YFP
  • GFP
  • CFP
  • RFP
  • pCS2 (+) from 22/06/12

Wednesday, 27th June

  1. MiniPrep- followed by nanodrop: confirmation of DNA presence in transformed bacteria
  2. Agarose gel electrophoresis (samples of 4 fluorescent DNA)
  3. Inoculation of colonies in LB medium- incubation at 37 degrees overnight

Thursday, 28th June

Gel migration 1

  • Gel preparation: 25mL agarose+TAE (freezer) + 2microL BET
  • Sample preparation:
    • DNA Ladder 1kB: 1microL ladder + 1microL loading buffer + 4microL ddH2O
    • Digested RFP, YFP, CFP, GFP, pCS2+ (from 28 june): 10microL sample + 2microL loading buffer
  • 100V, 1h
Result: no DNA detectable, Solution: to redo the step of digestion with more DNA

DNA digestion:

  • 4ul of CFP, GFP, RFP, YFP dosed on 28 june
  • 3ul of pSC2+ dosed on 28 june

Gel migration 2:

  • Gel preparation: 50mL agarose+TAE (freezer) + 4ul BET
  • Sample preparation:
    • DNA Ladder 1kB: 1ul ladder + 1ul loading buffer + 4ul ddH2O
    • Digested RFP, YFP, CFP, GFP, pCS2+ (from 29 june) : 15ul sample + 3ul loading buffer
  • 100V, 1h
Result: no RFP detectable (see picture below):

Friday, 29th June

Retrieved from "http://2012.igem.org/Team:Evry/Notebook/w3"