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Team:Groningen/protocols
From 2013.igem.org
| Line 62: | Line 62: | ||
<td>10ml</td> | <td>10ml</td> | ||
<td>1ml</td> | <td>1ml</td> | ||
| - | </tr></table> | + | </tr> |
| + | <tr> | ||
| + | <td>Ferric NH4 citrate(Sub4)</td> | ||
| + | <td>1ml</td> | ||
| + | <td>0,1ml</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Casein Hydrolysate</td> | ||
| + | <td>1g</td> | ||
| + | <td>0,1g</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Potassium Glutamate</td> | ||
| + | <td>2g</td> | ||
| + | <td>0,2g</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C | ||
| - | + | <br/>Sub3: Tri-Na Citrate 300mM | |
| - | + | <table border="1"> | |
| - | + | <tr> | |
| - | + | <td>Tri-Na Citrate</td> | |
| - | + | <td>0,8823g</td> | |
| - | + | </tr> | |
| - | Sub3: Tri-Na Citrate 300mM | + | <tr> |
| - | Tri-Na Citrate | + | <td>H2O</td> |
| - | H2O | + | <td>10ml</td> |
| + | </tr> | ||
| + | </table> | ||
Wrap in aluminum | Wrap in aluminum | ||
| - | Sub4: Ferric NH4 citrate | + | <br/>Sub4: Ferric NH4 citrate |
| - | Ferric NH4 citrate | + | <table border="1"> |
| - | H2O | + | <tr> |
| + | <td>Ferric NH4 citrate</td> | ||
| + | <td>0,22g</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>H2O</td> | ||
| + | <td>10ml</td> | ||
| + | </tr> | ||
| + | </table> | ||
Wrap in aluminum | Wrap in aluminum | ||
Revision as of 14:44, 17 July 2013
B. subtilis transformation
Losick protocol
-1 Day: Streak out strain and incubate plate o/n at 37 °C.
Transformation (D-Day):
1. Pick up a nice big colony and drop it in 2 ml of completed MC (1x)(see sub1).
2. Grow at 37 °C for 5 hours (more if culture is not really turbid).
3. Mix 400 µl of culture with DNA* in fresh tube (i.e. 15 ml tubes loosely closed – aeration) (*usually 1 µl. Then 10 µl of Quiagen plasmid miniprep or <1 µl of chromosal prep)
4. Grow for an additional 2 hours at 37 °C.
5. Plate all on selective antibiotic plates, and incubates at 37°C o/n.
Sub1: Copmpetence medium (MC completed)
| H20 | 1,8 ml | |
| 10x MC (sub2) | 200 ul | filter sterilize |
| MgSO4 | 6,7 ul | autoclave sterilize |
| Tryptophan 1% | 10 ul | filter steralize, wrap in Al |
Sub2: MC 10x
| for | 100 ml | 10 ml |
| K2H PO4 | 14,036g | 1,4036g |
| KH2 PO$ | 5,239g | 0,5239g |
| Glucose | 20g | 2g |
| Tri-Na Citrate 300mM(Sub3) | 10ml | 1ml |
| Ferric NH4 citrate(Sub4) | 1ml | 0,1ml |
| Casein Hydrolysate | 1g | 0,1g |
| Potassium Glutamate | 2g | 0,2g |
Add 50ml H2O, Mix, add H2O till 100ml, Filter sterilize and freeze at -20 °C
Sub3: Tri-Na Citrate 300mM
| Tri-Na Citrate | 0,8823g |
| H2O | 10ml |
Wrap in aluminum
Sub4: Ferric NH4 citrate
| Ferric NH4 citrate | 0,22g |
| H2O | 10ml |
Wrap in aluminum
