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Team:Bordeaux/NotebookJune23
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(Created page with "{{:Team:Bordeaux/Tete}} <html> <h1>19<sup>th</sup> June </h1> <div class="type-exp"> Molecular biology</div> <br> <ul class="day-detail"> <li>PCR of ELP-40 and ELP-60<br> <div...") |
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<div class="type-exp"> Molecular biology</div> | <div class="type-exp"> Molecular biology</div> | ||
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<li>PCR of ELP-40 and ELP-60<br> | <li>PCR of ELP-40 and ELP-60<br> | ||
| - | <div style="margin-left:95px ;"> => Dilution primer 100µM => 10µM | + | <div style="margin-left:95px ;"> => Dilution primer 100µM => 10µM<br> |
| - | Primer forward: 1µl primer + 9µl H2O (Tm=58°C) | + | Primer forward: 1µl primer + 9µl H2O (Tm=58°C)<br> |
| - | Primer reverse: 1µl primer + 9µl H2O (Tm=68°C)</div><br> | + | Primer reverse: 1µl primer + 9µl H2O (Tm=68°C)</div> |
| + | <br> | ||
| - | Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C <br> | + | Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C <br><br> |
Mix for 11 tubes</li><br> | Mix for 11 tubes</li><br> | ||
<table> | <table> | ||
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</table><br> | </table><br> | ||
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Thermal cycler : <br> | Thermal cycler : <br> | ||
| - | <div style="margin-left: | + | <div style="margin-left:55px ;"> |
3min 95°C<br> | 3min 95°C<br> | ||
30sec 95°C </div><br> | 30sec 95°C </div><br> | ||
25 cycles | 25 cycles | ||
| - | <div style="margin-left: | + | <div style="margin-left:55px ;"> |
30sec at all temperatures tested<br> | 30sec at all temperatures tested<br> | ||
3sec 72°C<br> | 3sec 72°C<br> | ||
5min 72°C</div> | 5min 72°C</div> | ||
</div> | </div> | ||
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<br> | <br> | ||
| + | |||
<ul class="day-detail"> | <ul class="day-detail"> | ||
<li>Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)<br> | <li>Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)<br> | ||
Revision as of 23:35, 17 October 2014
23rd June
Molecular biology
- PCR of ELP-40 and ELP-60
=> Dilution primer 100µM => 10µM
Primer forward: 1µl primer + 9µl H2O (Tm=58°C)
Primer reverse: 1µl primer + 9µl H2O (Tm=68°C)
Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C
Mix for 11 tubes - Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)
Digestion plasmid Digestion insert H2O 7,5µl 7,5µl 10X buffer 4 2µl 2µl XbaI 0,25µl 0,25µL EcoRI 0,25µl 0,25µl DNA 10µl 10µl - Digestion vector psB6A1 :
Digestion plasmid H2O 7,5µl 10X buffer 4 2µl XbaI 0,25µl EcoRI 0,25µl DNA 10µl - Digestion psB1C3 and PCR 40 and 60
OD PCR tube 40= 282,5 ng/μl
OD PCR tube 60= 366,3 ng/μl
Tube 40 = 0,88 DNA +9,12µl H2O
Tube 60 = 2,5 DNA + 7,5 µl H2O
| H2O | 357,5 µl |
| Buffer 5X | 110 µl |
| Primer | 27,5 µl |
| dNTP | 11µl |
Thermal cycler :
25 cycles
3min 95°C
30sec 95°C
30sec 95°C
25 cycles
30sec at all temperatures tested
3sec 72°C
5min 72°C
3sec 72°C
5min 72°C
With DNA = 6μl vector + 4μl H2O
With DNA= 0.56μl Miniprep product + 9,44μl H2O
Production
We want to do a preculture test to have a better production.
