Difference between revisions of "Team:Ruperto Carola/Experiments"

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<h2>LB-Medium</h2>
 
<h2>LB-Medium</h2>
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   </tr>
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</table>
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<h2>Synthetic complete (SC)</h2>
 +
<table style>
 +
  <tr>
 +
    <th>Component</th>
 +
    <th>Final concentration</th>
 +
  </tr>
 +
  <tr>
 +
    <td> Bacto yeast nitrogen base without amino acids</td>
 +
    <td>6.7 g/l</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Amino-acid (dropout) mix </td>
 +
    <td>2 g/l</td>
 +
  </tr>
 +
<tr>
 +
    <td>Glucose</td>
 +
    <td>2% (w/v)</td>
 +
  </tr>
 +
<tr>
 +
    <td>Bacto-agar (for solid media agar plates)</td>
 +
    <td>20 g/l</td>
 +
  </tr>
 +
</table>
 +
 +
 +
<h2>Composition of HiFi PCR</h2>
 +
 +
<table style>
 +
  <tr>
 +
    <th>Component</th>
 +
    <th>Final concentration</th>
 +
  </tr>
 +
  <tr>
 +
    <td> HiFi-buffer</td>
 +
    <td>x1</td>
 +
  </tr>
 +
  <tr>
 +
    <td>dNTPs</td>
 +
    <td>1 mM</td>
 +
  </tr>
 +
<tr>
 +
    <td>MgCl2</td>
 +
    <td>2.5 mM (kanMX6) or 3.5 mM (natNT2)</td>
 +
  </tr>
 +
<tr>
 +
    <td>Primer FWD or REV</td>
 +
    <td>100 ng</td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>Betaine</td>
 +
    <td>0.5 M</td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>HiFi-polymerase</td>
 +
    <td>0.5 M</td>
 +
  </tr>
 +
 +
</table>
 +
 +
 +
<h2>HiFi PCR cycling parameters</h2>
 +
 +
<table style>
 +
  <tr>
 +
    <th>Step</th>
 +
    <th>Temperature</th>
 +
<th>Time</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Initiation</td>
 +
    <td>97°C</td>
 +
<td>3 sec</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Denaturation</td>
 +
    <td>97°C </td>
 +
<td>20 sec</td>
 +
  </tr>
 +
<tr>
 +
    <td>Annealing</td>
 +
    <td>66°C</td>#
 +
<td>30 sec</td>
 +
  </tr>
 +
<tr>
 +
    <td>Elongation</td>
 +
    <td>72°C</td>
 +
<td>45 sec/kb</td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>Cycling: repeat steps 2-4 x29</td>
 +
    <td></td>
 +
<td></td>
 +
  </tr>
 +
 +
<tr>
 +
    <td>Final elongation</td>
 +
    <td>72°C</td>
 +
    <td>5 min</td>
 +
  </tr>
 +
<tr>
 +
    <td>Hold</td>
 +
    <td>4°C</td>
 +
    <td>∞</td>
 +
  </tr>
 +
  
 
</table>
 
</table>
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       <h2>TE</h2>
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       <h2>TAE</h2>
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<table>
 
<table>
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</table>
 
</table>
  
−
Reagent Volume
 
−
Final concentration
 
−
1M Tris-Cl (pH 8.0) 1 mL 10 mM
 
−
0.5M EDTA (pH 8.0) 0.2 mL 1 mM
 
−
Distilled H2O 98.8 mL
 
 
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 +
<h2>LiSORB</h2>
 +
<table>
 +
  <tr>
 +
    <th>Component</th>
 +
    <th>Final concentration</th>
 +
  </tr>
 +
  <tr>
 +
    <td> LiOAc</td>
 +
    <td>100 mM</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Tris HCl</td>
 +
    <td>10 mM</td>
 +
  </tr>
 +
<tr>
 +
    <td>EDTA (pH 8.0 with NaOH)</td>
 +
    <td>1 mM</td>
 +
  </tr>
 +
<tr>
 +
    <td>Sorbitol</td>
 +
    <td>1 M</td>
 +
  </tr>
 +
<tr>
 +
    <td>Salmon sperm DNA (10 mg/ml)</td>
 +
    <td>4 mg/ml</td>
 +
  </tr>
 +
</table>
 +
 +
<h2>LiPEG</h2>
 +
 +
<table style="width:100%">
 +
  <tr>
 +
    <th>Component</th>
 +
    <th>Final concentration</th>
 +
  </tr>
 +
  <tr>
 +
    <td> LiOAc</td>
 +
    <td>100 mM</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Tris HCl</td>
 +
    <td>10 mM</td>
 +
  </tr>
 +
<tr>
 +
    <td>EDTA (pH 8.0 with NaOH)</td>
 +
    <td>1 mM</td>
 +
  </tr>
 +
<tr>
 +
    <td>Polyethylene glycol (PEG) 3350 </td>
 +
    <td>40%</td>
 +
  </tr>
 +
</table>
 +
 +
 
PCR
 
PCR
 +
 
Restriction digest
 
Restriction digest
 
Yeast and bacterial transformation
 
Yeast and bacterial transformation
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<table>
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  <tr>
 +
    <th>Name F</th>
 +
    <th>Name R</th>
 +
    <th>Sequence F</th>
 +
    <th>Sequence R</th>
 +
  </tr>
 +
  <tr>
 +
    <td>STE2 rev  overhang 415</td>
 +
    <td>STE2 for overhang 415</td>
 +
    <td>agggaacaaagctggagctccaccgcggtggcggccgctctagaactagtggatccd</t>
 +
    <td>accgggccccccctcgaggtcgacggtatcgataagcttgatatcgaattcctgcag</t>
 +
  </tr>
 +
  <tr>
 +
    <td>S3 knock-out</td>
 +
    <td>S2 knock-out</td>
 +
    <td> ACAGGAAAATGAGTCATTGAGATCGAAACTTTTCAACCTATCAATCAACAATCCCCGTACGCTGCAGGTCGAC </t>
 +
    <td>AGAGAGTGACTGAGTGTTACATTAAATATATTTATATATAAACGTATGATATTTAATCGATGAATTCG</t>
 +
  </tr>
 +
  <tr>
 +
    <td>STE2 forBamh1</td>
 +
    <td>STE2 for Bam H1 </td>
 +
    <td> TTTTTGGATCCATGTCTGATGCGGCTCCTTC</t>
 +
    <td>TCGAGGGGGGGCCCGGTACCCTTATTATTATCTTCAGTCCAG</t>
 +
  </tr>
 +
</table>
 +
 
         Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.
 
         Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.
 
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Revision as of 03:43, 22 October 2019

Protocols


LB-Medium

Chemical amount in [g/l]
yeastextrakt 5
pepton/ trypton 10
NaCl 10

Synthetic complete (SC)

Component Final concentration
Bacto yeast nitrogen base without amino acids 6.7 g/l
Amino-acid (dropout) mix 2 g/l
Glucose 2% (w/v)
Bacto-agar (for solid media agar plates) 20 g/l

Composition of HiFi PCR

Component Final concentration
HiFi-buffer x1
dNTPs 1 mM
MgCl2 2.5 mM (kanMX6) or 3.5 mM (natNT2)
Primer FWD or REV 100 ng
Betaine 0.5 M
HiFi-polymerase 0.5 M

HiFi PCR cycling parameters

#
Step Temperature Time
Initiation 97°C 3 sec
Denaturation 97°C 20 sec
Annealing 66°C30 sec
Elongation 72°C 45 sec/kb
Cycling: repeat steps 2-4 x29
Final elongation 72°C 5 min
Hold 4°C ∞

TAE

Reagent Volume Final concentration
1M Tris-Cl (pH 8.0) 1 nL 10 mM
0.5M EDTA (pH 8.0) 0.2 mL 1 mM
Distilled H2O 98.8 mL

LiSORB

Component Final concentration
LiOAc 100 mM
Tris HCl 10 mM
EDTA (pH 8.0 with NaOH) 1 mM
Sorbitol 1 M
Salmon sperm DNA (10 mg/ml) 4 mg/ml

LiPEG

Component Final concentration
LiOAc 100 mM
Tris HCl 10 mM
EDTA (pH 8.0 with NaOH) 1 mM
Polyethylene glycol (PEG) 3350 40%
PCR Restriction digest Yeast and bacterial transformation Gel electrophoresis

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Name F Name R Sequence F Sequence R
STE2 rev overhang 415 STE2 for overhang 415 agggaacaaagctggagctccaccgcggtggcggccgctctagaactagtggatccd accgggccccccctcgaggtcgacggtatcgataagcttgatatcgaattcctgcag
S3 knock-out S2 knock-out ACAGGAAAATGAGTCATTGAGATCGAAACTTTTCAACCTATCAATCAACAATCCCCGTACGCTGCAGGTCGAC AGAGAGTGACTGAGTGTTACATTAAATATATTTATATATAAACGTATGATATTTAATCGATGAATTCG
STE2 forBamh1 STE2 for Bam H1 TTTTTGGATCCATGTCTGATGCGGCTCCTTC TCGAGGGGGGGCCCGGTACCCTTATTATTATCTTCAGTCCAG
Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

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You opened the first week and this last one, I guess? Great, so as you might know: this one is the week after wiki freeze, so the lab will transform into a boilerroom similiar to the sponsoring phase. Its selling time again. $$$