Team:Goettingen/notebook materials
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<p> | <p> | ||
<b>Antibiotics</b><br /> | <b>Antibiotics</b><br /> | ||
- | Ampicillin 100 µg/ | + | |
- | Gentamicin 10 µg/ | + | <center><table class="table1" style="width:400px"> |
- | Kanamycin 50 µl/ | + | <tr style="font-weight:bold;"> |
- | Chloramphenicol | + | <th style="font-size:80%;">Name</th> |
+ | <th style="font-size:80%;">Concentration</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>Ampicillin</td><td>100 µg/ml</td></tr> | ||
+ | <tr><td>Gentamicin</td><td>10 µg/ml</td></tr> | ||
+ | <tr><td>Kanamycin</td><td>50 µl/ml</td></tr> | ||
+ | <tr><td>Chloramphenicol</td><td>15 µg/ml</td></tr> | ||
+ | </table></center><br /><br /> | ||
<b>Supplements</b><br /> | <b>Supplements</b><br /> | ||
- | Tryptophane 50 mg/l< | + | <center><table class="table1" style="width:400px"> |
- | Histidine 20 mg/ | + | <tr style="font-weight:bold;"> |
- | Leucine 100 mg/ | + | <th style="font-size:80%;">Name</th> |
- | Uracile 20 mg/ | + | <th style="font-size:80%;">Concentration</th> |
- | Adenine 100 mg/ | + | |
- | + | </tr> | |
+ | |||
+ | <tr><td>Tryptophane</td><td>50 mg/l</td></tr> | ||
+ | <tr><td>Histidine</td><td>20 mg/l</td></tr> | ||
+ | <tr><td>Leucine</td><td>100 mg/l</td></tr> | ||
+ | <tr><td>Uracile</td><td>20 mg/l</td></tr> | ||
+ | <tr><td>Adenine</td><td>100 mg/l</td></tr> | ||
+ | <tr><td>3-amino-1,2,4-triazole (3-AT)</td><td>3-50 mM</td></tr> | ||
+ | </table></center><br /><br /> | ||
<b>LB medium</b><br /> | <b>LB medium</b><br /> | ||
For 1 liter:</br> | For 1 liter:</br> | ||
- | 10 g | + | |
- | + | <center><table class="table1" style="width:400px"> | |
- | 10 g | + | <tr style="font-weight:bold;"> |
- | < | + | <th style="font-size:80%;">Reagent</th> |
- | + | <th style="font-size:80%;">Amount</th> | |
+ | |||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr><td>Tryptone</td><td>10 g</td></tr> | ||
+ | <tr><td>Yeast Extract</td><td>5 g</td></tr> | ||
+ | <tr><td>NaCl</td><td>10 g</td></tr> | ||
+ | <tr><td colspan="3">add if needed:</td></tr> | ||
+ | <tr><td>Agar-agar</td><td>15 g</td></tr></table></center><br /><br /> | ||
<b>YPD medium</b><br /> | <b>YPD medium</b><br /> | ||
- | + | For 1 liter:<br /> | |
- | 20 g | + | <center><table class="table1" style="width:500px"> |
- | + | <tr style="font-weight:bold;"> | |
- | + | <th style="font-size:80%;">Reagent</th> | |
- | <i>or</i> after autoclaving 50 | + | <th style="font-size:80%;">Amount</th> |
- | add | + | </tr> |
- | + | ||
+ | <tr><td>Tryptone</td><td>20 g</td></tr> | ||
+ | <tr><td>Yeast Extract</td><td>10 g</td></tr> | ||
+ | <tr><td>Glucose</td><td>20 g | ||
+ | <i>or</i> after autoclaving 50 ml of 40 % Glucose-solution</td></tr> | ||
+ | <tr><td colspan="3">add if needed:</td></tr> | ||
+ | <tr><td>Agar-agar</td><td>15 g</td></tr></table></center><br /><br /> | ||
<b>YPAD medium</b><br /> | <b>YPAD medium</b><br /> | ||
Line 54: | Line 85: | ||
<b>50 fold ASPA solution</b><br /> | <b>50 fold ASPA solution</b><br /> | ||
For 1 liter:<br /> | For 1 liter:<br /> | ||
- | 297.45 g | + | <center><table class="table1" style="width:400px"> |
- | 26.09 g | + | <tr style="font-weight:bold;"> |
- | + | <th style="font-size:80%;">Reagent</th> | |
- | + | <th style="font-size:80%;">Amount</th> | |
+ | |||
+ | </tr> | ||
+ | <tr><td>NaNO<sub>3</sub></td><td>297.45 g</td></tr> | ||
+ | <tr><td>KCl</td><td>26.09 g</td></tr> | ||
+ | <tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>76.21 g</td></tr> | ||
+ | <tr><td colspan="3">pH 5.5</td></tr></table></center><br /><br /> | ||
<b>1000 fold Trace elements solution</b><br /> | <b>1000 fold Trace elements solution</b><br /> | ||
- | For 1 liter: < | + | For 1 liter: |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>ZnSO<sub>4</sub><td>12.27 g</td></tr> | ||
+ | <tr><td>H<sub>3</sub>Bo<sub>3</sub><td>11.01 g</td></tr> | ||
+ | <tr><td>FeSO<sub>4</sub><td>2.73 g</td></tr> | ||
+ | <tr><td>MnCl<sub>2</sub><td>3.15 g</td></tr> | ||
+ | <tr><td>CoCl<sub>2</sub><td>0.922 g</td></tr> | ||
+ | <tr><td>CuSO<sub>4</sub><td>1.022 g</td></tr> | ||
+ | <tr><td>Na<sub>2</sub>MoO<sub>4</sub></td><td>1.277 g </td></tr> | ||
+ | <tr><td>Na<sub>2</sub>EDTA</td><td>64.77 g </tr></table></center><br /><br /> | ||
<b>Minimal medium for<i> Aspergillus</i> cultivation</b><br /> | <b>Minimal medium for<i> Aspergillus</i> cultivation</b><br /> | ||
- | For 1 liter:<br /> | + | For 1 liter:<br /> |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>Glucose 40 %<td>25 ml</td></tr> | ||
+ | <tr><td>Trace elements 1000x</td><td>1 ml</td></tr> | ||
+ | <tr><td>ASPA 50x</td> <td>20 ml</td></tr> | ||
+ | <tr><td>MgSO<sub>4</sub> 1 M</td><td>2 ml</td></tr></table></center><br /><br /> | ||
+ | <tr><td colspan="3">add if needed:</td></tr> | ||
+ | <tr><td>Agar-agar</td><td>15 g</td></tr></table></center><br /><br /> | ||
+ | |||
+ | |||
<b>50 fold TAE buffer</b><br /> | <b>50 fold TAE buffer</b><br /> | ||
- | For 1 liter: <br />242 g | + | For 1 liter: <br /> |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>Tris</td><td>242 g</td></tr> | ||
+ | <tr><td>EDTA (0.5 M; pH 8.0)</td><td>100 ml</td></tr> | ||
+ | <tr><td colspan="3">dissolve in 60 % of final volume</td></tr> | ||
+ | <tr><td colspan="3">add 57.1 mL acetic acid (100 %) and fill up to 1 liter.</td></tr></table></center> | ||
<br /><br /> | <br /><br /> | ||
<b>SC dropout medium </b><br /> | <b>SC dropout medium </b><br /> | ||
- | + | <center><table class="table1" style="width:400px"> | |
- | + | <tr style="font-weight:bold;"> | |
- | + | <th style="font-size:80%;">Name</th> | |
- | + | <th style="font-size:80%;">Amount</th> | |
- | + | ||
+ | </tr> | ||
+ | <tr><td>Yeast Nitrogen Base (without amino acids)</td><td>6.7 g</td></tr> | ||
+ | <tr><td>Amino acid mixture (-Leu/-Trp/-His/-Ura)</td><td>1.46 g</td></tr> | ||
+ | <tr><td colspan="3">Adjust pH to 5.6</td></tr> | ||
+ | <tr><td colspan="3">Autoclave 15 min only at 121°C</td></tr> | ||
+ | <tr><td colspan="3">Add 50 ml sterile Glucose (40%), possible to add 3-50 ml 3-AT</td></tr></table></center><br /><br /> | ||
<b>SOB medium </b><br /> | <b>SOB medium </b><br /> | ||
- | + | For 1 liter: <br /> | |
- | + | <center><table class="table1" style="width:400px"> | |
- | + | <tr style="font-weight:bold;"> | |
- | + | <th style="font-size:80%;">Name</th> | |
- | + | <th style="font-size:80%;">Amount</th> | |
- | + | ||
- | + | ||
- | + | ||
+ | </tr> | ||
+ | |||
+ | <tr><td>Tryptone</td><td>20 g</td></tr> | ||
+ | <tr><td>Yeast extract</td> <td>5 g</td></tr> | ||
+ | <tr><td>NaCl</td><td>0.584 g</td></tr> | ||
+ | <tr><td>KCl</td><td>0.186 g</td></tr> | ||
+ | <tr><td colspan="2">add dH2O to 1000 ml</td></tr> | ||
+ | <tr><td>MgCl<sub>2</sub></td><td>2.032 g</td></tr> | ||
+ | <tr><td>MgSO<sub>4</sub></td><td>2.064 g</td></tr> | ||
+ | <tr><td colspan="2">pH with NaOH to 7.0</td></tr></table></center> | ||
+ | |||
+ | <br /><br /> | ||
+ | |||
+ | </p> | ||
<h2>Materials & Methods for yeast protocols</h2> | <h2>Materials & Methods for yeast protocols</h2> | ||
- | <b>AA-mixture</b><br /> | + | <p><b>AA-mixture</b><br /> |
- | For 1 liter (selection): <br />Arginine 50 mg | + | For 1 liter (selection): <br /> |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | |||
+ | <tr><td>Arginine</td><td>50 mg</td></tr> | ||
+ | <tr><td>Aspartic Acid</td><td>80 mg</td></tr> | ||
+ | <tr><td>Histidine</td><td>20 mg</td></tr> | ||
+ | <tr><td>Isoleucine</td><td>50 mg</td></tr> | ||
+ | <tr><td>Leucine</td><td>100 mg</td></tr> | ||
+ | <tr><td>Lysine</td><td>50 mg</td></tr> | ||
+ | <tr><td>Methionine</td><td>20 mg</td></tr> | ||
+ | <tr><td>Phenylalanine</td><td>50 mg</td></tr> | ||
+ | <tr><td>Threonine</td><td>100 mg</td></tr> | ||
+ | <tr><td>Tryptophan</td><td>50 mg</td></tr> | ||
+ | <tr><td>Tyrosine</td><td>50 mg</td></tr> | ||
+ | <tr><td>Uracil</td><td>20 mg</td></tr> | ||
+ | <tr><td>Valine</td><td>140 mg</td></tr> | ||
+ | <tr><td>Serine</td><td>20 mg</td></tr> | ||
+ | <tr><td>Adenine</td><td>120 mg</td></tr> | ||
+ | <tr><td>Histidine</td><td>20mg</td></tr> | ||
+ | </table></center><br /><br /> | ||
+ | |||
<b>Synthetic complete dropout (SC/SD) medium</b><br /> | <b>Synthetic complete dropout (SC/SD) medium</b><br /> | ||
- | For 1 liter: <br /> | + | For 1 liter: <br /> |
+ | <center><table class="table1" style="width:600px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>Yeast nitrogen base (YNB) without amino acids</td><td>6.7 g</td></tr> | ||
+ | |||
+ | <tr><td colspan='2'>If needed:</td></tr> | ||
+ | <tr><td>Bacto-Agar (autoclave only 15 min)</td><td>18 g</td></tr> | ||
+ | <tr><td colspan='2'>add. 50 ml of 40 % sterile glucose and recommended amino acids after autoclaving.</td></tr> | ||
+ | </table></center> <br /><br /> | ||
<b>SORB </b><br /> | <b>SORB </b><br /> | ||
- | For 1 liter: <br />6.59 g | + | For 1 liter: <br /> |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>LiAC</td><td>6.59 g</td></tr> | ||
+ | <tr><td>Tris<td>1.21 g</td></tr> | ||
+ | <tr><td colspan='2'>adj. pH with HCl to 8.0;</td></tr> | ||
+ | <tr><td colspan='2'>add 0.37 g 2NaH2EDTA</td></tr> | ||
+ | <tr><td>Sorbitol</td><td>182.2 g</td></tr> | ||
+ | <tr><td colspan='2'>pH 8.0 with acetic acid</td></tr> | ||
+ | </table></center> | ||
+ | |||
+ | <br /><br /> | ||
<b>LIT-PEG</b><br /> | <b>LIT-PEG</b><br /> | ||
- | For 1 liter: < | + | For 1 liter: |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr><td>LiAC</td><td>6.59 g </td></tr> | ||
+ | <tr><td>Tris</td><td>1.21 g </td></tr> | ||
+ | <tr><td>2NaH2EDTA</td><td>0.37 g </td></tr> | ||
+ | <tr><td>PEG 4000 (prepare 10 mL prior to use)</td><td>400 g </td></tr> | ||
+ | </table></center> | ||
+ | <br /><br /> | ||
<b>TE buffer </b><br /> | <b>TE buffer </b><br /> | ||
- | For 1 liter: <br /> | + | For 1 liter: <br /> |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
- | <b>5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal-stock, 500x)</b><br /> | + | </tr> |
- | For 5 mL:< | + | <tr><td>Tris</td><td>0.12 g </td></tr> |
- | Store X-α-Gal solutions at -20°C in the dark.<br /><br /> | + | <tr><td>2NaH2EDTA</td><td>0.37 g </td></tr> |
+ | </table></center> | ||
+ | <br /><br /> | ||
+ | |||
+ | <b>5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal-stock, 500x)</b><br | ||
+ | |||
+ | /> | ||
+ | For 5 mL: | ||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td colspan='3'>dissolve 100 mg X-α-Gal in dimethylformamide (DMF; toxic).</td></tr> | ||
+ | <tr><td colspan='3'>Store X-α-Gal solutions at -20°C in the dark.</td></tr> | ||
+ | </table></center> | ||
+ | <br /><br /> | ||
<b>3-amino-1,2,4-triazole (3-AT, 1 M)</b><br /> | <b>3-amino-1,2,4-triazole (3-AT, 1 M)</b><br /> | ||
- | For 50 mL:< | + | For 50 mL: |
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | </tr> | ||
+ | <tr><td colspan='3'>Dissolve 4.2 g 3-AT in sterile water.</td></tr> | ||
+ | <tr><td colspan='3'>Store at 4°C in the dark.</td></tr> | ||
+ | </table></center> | ||
+ | |||
+ | <br | ||
+ | |||
+ | /><br /> | ||
+ | |||
+ | </p> | ||
<h2>Materials & Methods for <i>E.coli</i> protocols</h2> | <h2>Materials & Methods for <i>E.coli</i> protocols</h2> | ||
+ | <p> | ||
<b>SOB-medium (super optimal broth)</b> | <b>SOB-medium (super optimal broth)</b> | ||
- | For 1 liter: <br /> | + | For 1 liter: <br /> |
- | Adjust to pH 7.5 prior to use. < | + | <center><table class="table1" style="width:400px"> |
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>yeast extract</td><td>5 g </td></tr> | ||
+ | <tr><td>tryptone</td><td>20 g </td></tr> | ||
+ | <tr><td>NaCl</td><td>0.584 g </td></tr> | ||
+ | <tr><td>KCl</td><td>0.186 g </td></tr> | ||
+ | <tr><td>MgSO<sub>4</sub></td><td>2.4 g </td></tr> | ||
+ | <tr><td colspan='3'>Adjust to pH 7.5 prior to use.</td></tr> | ||
+ | <tr><td colspan='3'>This requires approximately 25 ml of 1M | ||
+ | |||
+ | NaOH per liter.</td></tr> | ||
+ | |||
+ | </table></center> | ||
+ | |||
+ | <br /><br /> | ||
<b>TB-buffer</b><br /> | <b>TB-buffer</b><br /> | ||
- | For 1 liter: <br />18.65 g | + | For 1 liter: <br /> |
- | Adjust pH to 6.7 with 1M KOH | + | <center><table class="table1" style="width:400px"> |
- | Add gradually 10.88 g manganese chloride to 100 ml water until it is dissolved | + | <tr style="font-weight:bold;"> |
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | |||
+ | <tr><td>potassium chloride</td><td>18.65 g </td></tr> | ||
+ | <tr><td>calcium chloride</td><td>2.2 g </td></tr> | ||
+ | <tr><td>0.5 M PIPES</td><td>20 ml </td></tr> | ||
+ | <tr><td colspan='2'>add 800 ml water</td></tr> | ||
+ | <tr><td colspan='2'>Adjust pH to 6.7 with 1M KOH</td></tr> | ||
+ | <tr><td colspan='2'>Add gradually 10.88 g manganese chloride to 100 ml water until it is dissolved</td></tr> | ||
+ | |||
+ | |||
+ | <tr><td colspan='2'>fill up to 1 liter. <br />Store at 4°C after sterile filtration.</td></tr> | ||
+ | |||
+ | </table></center> | ||
+ | <br /><br | ||
+ | |||
+ | /> | ||
<b>BD-solutions (100 mL each)</b><br /> | <b>BD-solutions (100 mL each)</b><br /> | ||
- | BD I: 0.9 g | + | BD I: |
- | BD II A: 16 g | + | <center><table class="table1" style="width:400px"> |
- | BD II B: 2 g | + | <tr style="font-weight:bold;"> |
- | + | <th style="font-size:80%;">Name</th> | |
- | BD III: 29.45 g | + | <th style="font-size:80%;">Amount</th> |
+ | |||
+ | </tr> | ||
+ | <tr><td>glucose</td><td>0.9 g </td></tr> | ||
+ | <tr><td>lysozyme</td><td>2 g </td></tr> | ||
+ | <tr><td colspan='3'>+ 10 μl RNase (10 mg/ml)/ 200 μl BD I</td></tr> | ||
+ | </table></center><br /><br /> | ||
+ | BD II A: | ||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>NaOH</td><td>16 g </td></tr> | ||
+ | <tr><td>EDTA </td><td>0.315 g </td></tr> | ||
+ | </table></center><br /><br /> | ||
+ | |||
+ | BD II B: | ||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>SDS</td><td>2 g </td></tr> | ||
+ | <tr><td>Tris (adjusted with HCl to pH 8 )</td><td>0.3 ml </td></tr> | ||
+ | <tr><td colspan='2'> → working solution: mix stock A & B in a 1:1 ratio</td></tr> | ||
+ | </table></center><br /><br /> | ||
+ | BD III: | ||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>potassium acetate</td><td>29.45 g </td></tr> | ||
+ | <tr><td>formic acid</td><td>5.4 mL </td></tr> | ||
+ | </table></center> | ||
+ | <br /><br /> | ||
<b>CCMB80 buffer</b><br /> | <b>CCMB80 buffer</b><br /> | ||
- | For 1 liter: < | + | For 1 liter: |
- | + | <center><table class="table1" style="width:400px"> | |
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
- | + | </tr> | |
+ | |||
+ | <tr><td>KOAc 1M stock pH 7.0</td><td>10 ml </td></tr> | ||
+ | <tr><td>CaCl<sub>2</sub> ∙ 2 H<sub>2</sub>O</td><td>11.8 g </td></tr> | ||
+ | <tr><td>MnCl<sub>2</sub> ∙ 4 H<sub>2</sub>O</td><td>4 g </td></tr> | ||
+ | <tr><td>MgCl<sub>2</sub> ∙ 6 H<sub>2</sub>O</td><td>2 g </td></tr> | ||
+ | <tr><td>glycerol 100 %</td><td>100 mL </td></tr> | ||
+ | <tr><td colspan='2'>Lower pH to 6.4 with 0.1 M HCl</td></tr> | ||
+ | <tr><td colspan='2'>- An increase in pH will result in | ||
+ | |||
+ | precipitation of manganese dioxide from Mn containing solutions</td></tr> | ||
+ | <tr><td colspan='3'>sterile filtration and store at 4°C.</td></tr> | ||
+ | </table></center> | ||
+ | </p><br /> | ||
+ | <h2>Materials & Methods for Protein Analysis</h2> | ||
+ | <p> | ||
+ | <b>5 x SDS Loading Dye</b><br /> | ||
+ | For 10 ml: <br /> | ||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>Glycerol (100%)</td><td>3 ml</td></tr> | ||
+ | <tr><td>SDS (20%)</td><td>2 ml </td></tr> | ||
+ | <tr><td>β-Mercaptoethanol (100%)</td><td>1.6 ml </td></tr> | ||
+ | <tr><td>H<sub>2</sub>O</td><td>2 ml </td></tr> | ||
+ | <tr><td>Tris-HCl (pH 7.0)</td><td>1.4 ml</td></tr> | ||
+ | |||
+ | </table></center> | ||
+ | <br /><br /> | ||
+ | |||
+ | |||
+ | <b>10 x PAGE buffer, pH 8.3</b><br /> | ||
+ | For 1 liter: <br /> | ||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | |||
+ | <tr><td>glycine</td><td>144.13 g </td></tr> | ||
+ | <tr><td>Tris</td><td>30.25 g </td></tr> | ||
+ | <tr><td>SDS</td><td>10 g </td></tr> | ||
+ | |||
+ | </table></center> | ||
+ | <br /><br /> | ||
+ | |||
+ | |||
+ | </p> | ||
+ | <br /> | ||
+ | <h2>Denaturing Polyacrylamid Gel Electrophoresis (PAGE) </h2> | ||
+ | <p> | ||
+ | <table class="table2"> | ||
+ | <tr><th>Running gel (12 %, 2 gels)</th><th>Stacking gel (5 %, 2 gels)</th></tr> | ||
+ | <tr><td>6.6 ml H2O</td><td>6.8 ml H2O</td></tr> | ||
+ | <tr><td>8.0 ml Bis-Acrylamid (37, 5:1)</td><td>1.7 ml Bis-Acrylamid (37, 5:1) | ||
+ | |||
+ | </td></tr> | ||
+ | <tr><td>5.0 ml 1.5 M Tris-HCl (pH 8.8)</td><td>1.25 ml 1.5 M Tris-HCl (pH 6.8) | ||
+ | |||
+ | </td></tr> | ||
+ | <tr><td>200 µl SDS (10 %)</td><td>100 µl SDS (10 %)</td></tr> | ||
+ | <tr><td>200 µl APS (10 %)</td><td>100 µl APS (10 %)</td></tr> | ||
+ | <tr><td>10 µl TEMED</td><td>10 µl TEMED</td></tr> | ||
+ | </table> | ||
+ | <br /> | ||
+ | <table class="table2"> | ||
+ | <tr><td><b>Fixation solution</b></td><td>10 % Acetic Acid - 45 % MetOH</td></tr> | ||
+ | <tr><td><b>Staining solution</b></td><td>0.5 % Coomassie Brilliant Blue - 10 % | ||
+ | |||
+ | Acetic Acid - 45 % MetOH</td></tr> | ||
+ | <tr><td><b>Destaining solution</b></td><td>10 % Acetic acid</td></tr> | ||
+ | </table> | ||
+ | <br /> | ||
+ | </p> | ||
+ | <p> | ||
+ | <b>10 x Buffer W </b><br /> | ||
+ | For 1 liter: | ||
+ | |||
+ | <center><table class="table1" style="width:400px"> | ||
+ | <tr style="font-weight:bold;"> | ||
+ | <th style="font-size:80%;">Name</th> | ||
+ | <th style="font-size:80%;">Amount</th> | ||
+ | |||
+ | </tr> | ||
+ | <tr><td>Tris/HCl pH 8.0</td><td>6.05 g </td></tr> | ||
+ | <tr><td>NaCl</td><td>8.77 g </td></tr> | ||
+ | <tr><td>Na<sub>2</sub> ∙ EDTA</td><td>0.37 g </td></tr> | ||
+ | |||
+ | </table></center> | ||
+ | <br /><br /> | ||
+ | |||
+ | |||
+ | <b>Buffer E</b><br /> | ||
+ | For 100 ml: use 100 ml buffer w and add 0.536 g D-desthiobiotin<br /><br /> | ||
+ | <b>Buffer R</b><br /> | ||
+ | For 100 ml: use 100 ml buffer w and add 0,242 g HABA (hydroxy-azophenyl-benzoic acid) pH 8.0<br /> | ||
+ | <br /> | ||
+ | <b>Isopropyl-β-D-thiogalactopyranosid (IPTG; 1M)</b><br /> | ||
+ | For 10 ml 1 M solution, weight 2.383 g IPTG and dissolve in water. <br /> | ||
+ | Make aliquots and store at -20°C after sterile filtration. <br /> | ||
+ | End concentration for induction is 1 mM.<br /><br /> | ||
+ | </p> | ||
+ | |||
+ | <h2>For <i>in vivo</i> tests </h2> | ||
+ | <p> | ||
+ | <b>PBS/<u>PBS+</u></b>:<br /><br /> | ||
+ | For 50 mL: 4 g NaCl, 0.1 g KCl, 0.72 g Na2HPO4, 0.12 g KH2PO4<br /> | ||
+ | Add optional: 0.0665 g<u> CaCl2 ∙ 2 H2O</u>, 0.05 g <u>MgCl2 ∙ 6 H2O</u><br /> | ||
+ | Adjust the pH to 7.4 (or 7.2) with HCl.<br /><br /> | ||
+ | <b>Calcofluor Fluorescent Brightener 28, Stock Solution (10 mg/ml)</b><br /><br /> | ||
+ | Dissolve 10 mg Calcofluor in 1 ml water, store in the dark and at -20°C.<br /> | ||
+ | </p> | ||
+ | <br /><br /> | ||
+ | <div><a href="/Team:Goettingen/notebook_wetlab/gfp" class="button_pre"><b>Previous</b></a></div> | ||
</div> | </div> | ||
+ | |||
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Latest revision as of 19:00, 17 October 2014
General Materials
Antibiotics
Name | Concentration |
---|---|
Ampicillin | 100 µg/ml |
Gentamicin | 10 µg/ml |
Kanamycin | 50 µl/ml |
Chloramphenicol | 15 µg/ml |
Supplements
Name | Concentration |
---|---|
Tryptophane | 50 mg/l |
Histidine | 20 mg/l |
Leucine | 100 mg/l |
Uracile | 20 mg/l |
Adenine | 100 mg/l |
3-amino-1,2,4-triazole (3-AT) | 3-50 mM |
LB medium
For 1 liter:
Reagent | Amount | |
---|---|---|
Tryptone | 10 g | |
Yeast Extract | 5 g | |
NaCl | 10 g | |
add if needed: | ||
Agar-agar | 15 g |
YPD medium
For 1 liter:
Reagent | Amount | |
---|---|---|
Tryptone | 20 g | |
Yeast Extract | 10 g | |
Glucose | 20 g or after autoclaving 50 ml of 40 % Glucose-solution | |
add if needed: | ||
Agar-agar | 15 g |
YPAD medium
prepare YPD medium and add 120 mg adenine.
50 fold ASPA solution
For 1 liter:
Reagent | Amount | |
---|---|---|
NaNO3 | 297.45 g | |
KCl | 26.09 g | |
KH2PO4 | 76.21 g | |
pH 5.5 |
1000 fold Trace elements solution
For 1 liter:
Name | Amount |
---|---|
ZnSO4 | 12.27 g |
H3Bo3 | 11.01 g |
FeSO4 | 2.73 g |
MnCl2 | 3.15 g |
CoCl2 | 0.922 g |
CuSO4 | 1.022 g |
Na2MoO4 | 1.277 g |
Na2EDTA | 64.77 g |
Minimal medium for Aspergillus cultivation
For 1 liter:
Name | Amount |
---|---|
Glucose 40 % | 25 ml |
Trace elements 1000x | 1 ml |
ASPA 50x | 20 ml |
MgSO4 1 M | 2 ml |
50 fold TAE buffer
For 1 liter:
Name | Amount | |
---|---|---|
Tris | 242 g | |
EDTA (0.5 M; pH 8.0) | 100 ml | |
dissolve in 60 % of final volume | ||
add 57.1 mL acetic acid (100 %) and fill up to 1 liter. |
SC dropout medium
Name | Amount | |
---|---|---|
Yeast Nitrogen Base (without amino acids) | 6.7 g | |
Amino acid mixture (-Leu/-Trp/-His/-Ura) | 1.46 g | |
Adjust pH to 5.6 | ||
Autoclave 15 min only at 121°C | ||
Add 50 ml sterile Glucose (40%), possible to add 3-50 ml 3-AT |
SOB medium
For 1 liter:
Name | Amount |
---|---|
Tryptone | 20 g |
Yeast extract | 5 g |
NaCl | 0.584 g |
KCl | 0.186 g |
add dH2O to 1000 ml | |
MgCl2 | 2.032 g |
MgSO4 | 2.064 g |
pH with NaOH to 7.0 |
Materials & Methods for yeast protocols
AA-mixture
For 1 liter (selection):
Name | Amount |
---|---|
Arginine | 50 mg |
Aspartic Acid | 80 mg |
Histidine | 20 mg |
Isoleucine | 50 mg |
Leucine | 100 mg |
Lysine | 50 mg |
Methionine | 20 mg |
Phenylalanine | 50 mg |
Threonine | 100 mg |
Tryptophan | 50 mg |
Tyrosine | 50 mg |
Uracil | 20 mg |
Valine | 140 mg |
Serine | 20 mg |
Adenine | 120 mg |
Histidine | 20mg |
Synthetic complete dropout (SC/SD) medium
For 1 liter:
Name | Amount |
---|---|
Yeast nitrogen base (YNB) without amino acids | 6.7 g |
If needed: | |
Bacto-Agar (autoclave only 15 min) | 18 g |
add. 50 ml of 40 % sterile glucose and recommended amino acids after autoclaving. |
SORB
For 1 liter:
Name | Amount |
---|---|
LiAC | 6.59 g |
Tris | 1.21 g |
adj. pH with HCl to 8.0; | |
add 0.37 g 2NaH2EDTA | |
Sorbitol | 182.2 g |
pH 8.0 with acetic acid |
LIT-PEG
For 1 liter:
Name | Amount |
---|---|
LiAC | 6.59 g |
Tris | 1.21 g |
2NaH2EDTA | 0.37 g |
PEG 4000 (prepare 10 mL prior to use) | 400 g |
TE buffer
For 1 liter:
Name | Amount |
---|---|
Tris | 0.12 g |
2NaH2EDTA | 0.37 g |
5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal-stock, 500x)
For 5 mL:
Name | Amount | |
---|---|---|
dissolve 100 mg X-α-Gal in dimethylformamide (DMF; toxic). | ||
Store X-α-Gal solutions at -20°C in the dark. |
3-amino-1,2,4-triazole (3-AT, 1 M)
For 50 mL:
Name | Amount | |
---|---|---|
Dissolve 4.2 g 3-AT in sterile water. | ||
Store at 4°C in the dark. |
Materials & Methods for E.coli protocols
SOB-medium (super optimal broth)
For 1 liter:
Name | Amount | |
---|---|---|
yeast extract | 5 g | |
tryptone | 20 g | |
NaCl | 0.584 g | |
KCl | 0.186 g | |
MgSO4 | 2.4 g | |
Adjust to pH 7.5 prior to use. | ||
This requires approximately 25 ml of 1M NaOH per liter. |
TB-buffer
For 1 liter:
Name | Amount |
---|---|
potassium chloride | 18.65 g |
calcium chloride | 2.2 g |
0.5 M PIPES | 20 ml |
add 800 ml water | |
Adjust pH to 6.7 with 1M KOH | |
Add gradually 10.88 g manganese chloride to 100 ml water until it is dissolved | |
fill up to 1 liter. Store at 4°C after sterile filtration. |
BD-solutions (100 mL each)
BD I:
Name | Amount | |
---|---|---|
glucose | 0.9 g | |
lysozyme | 2 g | |
+ 10 μl RNase (10 mg/ml)/ 200 μl BD I |
BD II A:
Name | Amount |
---|---|
NaOH | 16 g |
EDTA | 0.315 g |
BD II B:
Name | Amount |
---|---|
SDS | 2 g |
Tris (adjusted with HCl to pH 8 ) | 0.3 ml |
→ working solution: mix stock A & B in a 1:1 ratio |
BD III:
Name | Amount |
---|---|
potassium acetate | 29.45 g |
formic acid | 5.4 mL |
CCMB80 buffer
For 1 liter:
Name | Amount | |
---|---|---|
KOAc 1M stock pH 7.0 | 10 ml | |
CaCl2 ∙ 2 H2O | 11.8 g | |
MnCl2 ∙ 4 H2O | 4 g | |
MgCl2 ∙ 6 H2O | 2 g | |
glycerol 100 % | 100 mL | |
Lower pH to 6.4 with 0.1 M HCl | ||
- An increase in pH will result in precipitation of manganese dioxide from Mn containing solutions | ||
sterile filtration and store at 4°C. |
Materials & Methods for Protein Analysis
5 x SDS Loading Dye
For 10 ml:
Name | Amount |
---|---|
Glycerol (100%) | 3 ml |
SDS (20%) | 2 ml |
β-Mercaptoethanol (100%) | 1.6 ml |
H2O | 2 ml |
Tris-HCl (pH 7.0) | 1.4 ml |
10 x PAGE buffer, pH 8.3
For 1 liter:
Name | Amount |
---|---|
glycine | 144.13 g |
Tris | 30.25 g |
SDS | 10 g |
Denaturing Polyacrylamid Gel Electrophoresis (PAGE)
Running gel (12 %, 2 gels) | Stacking gel (5 %, 2 gels) |
---|---|
6.6 ml H2O | 6.8 ml H2O |
8.0 ml Bis-Acrylamid (37, 5:1) | 1.7 ml Bis-Acrylamid (37, 5:1) |
5.0 ml 1.5 M Tris-HCl (pH 8.8) | 1.25 ml 1.5 M Tris-HCl (pH 6.8) |
200 µl SDS (10 %) | 100 µl SDS (10 %) |
200 µl APS (10 %) | 100 µl APS (10 %) |
10 µl TEMED | 10 µl TEMED |
Fixation solution | 10 % Acetic Acid - 45 % MetOH |
Staining solution | 0.5 % Coomassie Brilliant Blue - 10 % Acetic Acid - 45 % MetOH |
Destaining solution | 10 % Acetic acid |
10 x Buffer W
For 1 liter:
Name | Amount |
---|---|
Tris/HCl pH 8.0 | 6.05 g |
NaCl | 8.77 g |
Na2 ∙ EDTA | 0.37 g |
Buffer E
For 100 ml: use 100 ml buffer w and add 0.536 g D-desthiobiotin
Buffer R
For 100 ml: use 100 ml buffer w and add 0,242 g HABA (hydroxy-azophenyl-benzoic acid) pH 8.0
Isopropyl-β-D-thiogalactopyranosid (IPTG; 1M)
For 10 ml 1 M solution, weight 2.383 g IPTG and dissolve in water.
Make aliquots and store at -20°C after sterile filtration.
End concentration for induction is 1 mM.
For in vivo tests
PBS/PBS+:
For 50 mL: 4 g NaCl, 0.1 g KCl, 0.72 g Na2HPO4, 0.12 g KH2PO4
Add optional: 0.0665 g CaCl2 ∙ 2 H2O, 0.05 g MgCl2 ∙ 6 H2O
Adjust the pH to 7.4 (or 7.2) with HCl.
Calcofluor Fluorescent Brightener 28, Stock Solution (10 mg/ml)
Dissolve 10 mg Calcofluor in 1 ml water, store in the dark and at -20°C.