Team:Evry/Notebook/w4

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<h1>Weeks</h1>
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<h1>Week 4: 2nd July - 8th July</h1>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w1">1</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w2">2</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w3">3</a></td>
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  <td>4</td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w5">5</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w6">6</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w7">7</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w8">8</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w9">9</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w10">10</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w11">11</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w12">12</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w13">13</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w14">14</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w15">15</a></td>
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w16">16</a></td>
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<h1>Week 1: 2nd July - 8th July</h1>
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<h2>Monday, 2nd July</h2>
<h2>Monday, 2nd July</h2>
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Elution in two times 25 uL with water RNAse free instead of EB buffer.
Elution in two times 25 uL with water RNAse free instead of EB buffer.
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<h2>Friday, 6th July</h2>
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Latest revision as of 03:54, 27 September 2012

Weeks:

June July August September October November

Week 4: 2nd July - 8th July

Monday, 2nd July

  1. T10 bacteria transformation with DNA ligated on Friday (29/06)- protocol as before
  2. Transformed bacteria spreaded on Petri's plate- LB-agar-ampiciline- incubation 37 degrees Celsius, overnight
  3. SOC preparation:
    • 1,4g Hanahan's Broth
    • 0,18g Glucose
    • 50mL MiliQ water
filtrated on microfilter (if you have more important volume, autoclave it)

Tuesday, 3rd July

  1. Designing starters
  2. Weekly team meeting- discussion about work organization

Wednesday, 4th July

  1. Gel electrophoresis of DNA digests (from 28.06.) followed by cutting-of bands of interests (pCS2+ plasmid and XFP plasmids)
  2. Gel-extraction (Kit used: QIA quick gel extraction) followed by nano-drop measurements in order to get information about DNA concentration.
    Result: very low quantity of DNA. We realized that quantity of DNA used for digestion was too low (less than 2 micrograms of DNA/sample).
  3. DNA Digestion (of pCS2+, CFP, RFP, GFP, YFP). Protocol(Final volume of sample: 30 uL):
    • 3 uL Buffer 10x (choose a right buffer for each enzyme you use)
    • 1 uL Restriction Enzyme 1
    • 1 uL Restriction Enzyme 2
    • 2 ug DNA (calculate the volume which you need to add, depends of the DNA concentration)
    • c uL ddH2O (fill till 30 uL)
    • Incubation 3h at 37 degrees

Thursday, 5th July

Gel Extraction 1:

  1. Excise the DNA fragment from agarose gel
  2. Weight the gel slice
  3. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg = 100 uL)
    Type
    Gel weight (g)
    V QG (uL)
    V iso (uL)
    pCS2
    0,13
    390
    130
    GFP
    0,10
    300
    100
    YFP
    0,08
    240
    80
    CFP
    0,10
    300
    100
  4. Incubate at 50 degrees Celsius for 10 min to dissolve the gel
  5. Add 1 gel volume of isopropanol to the sample and mix
  6. Apply the sample to the QIAquick column to bond DNA
  7. Centrifuge at 13 000 rpm for 1 min and discard flow-through
  8. Add 500 uL of Buffer QG to the column
  9. Centrifuge at 13 000 rpm for 1 min and discard flow-through
  10. To wash, add 750 uL of Buffer PE
  11. Centrifuge at 13 000 rpm for 1 min and discard flow-through
  12. Centrifuge the column again at 13 000 rpm for 1 min and place the column in a clean 1.5 mL tube
  13. Add 50 uL of Buffer EB to elute DNA
  14. Check DNA concentration with Nanodrop
DNA Concentration
Type
Concentration (ng.uL-1)
pCS2
9,4
GFP
3,1
YFP
4,7
CFP
4,7
But: Concentrations are really low => Need to optimize the protocol and make a new gel migration.

Gel Migration:

  • Gel at 0,08%
  • V tae = 30 mL
  • m aga = 0,24 g
  • V bet = 3 uL
No Band for RFP

Gel Extraction 2

Type
Gel weight (g)
V QG (uL)
V iso (uL)
pCS2
0,084
252
84
GFP
0,040
120
40
YFP
0,023
69
23
CFP
0,050
150
50
Protocole modification: Elution in two times 25 uL with water RNAse free instead of EB buffer.

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