IGEM:Groningen/Notebook/iGEM 2011/2011/07/12: Difference between revisions

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==Entry title==
==12-07-11==
* Insert content here...
<br> Very few colonies were seen on the plates of yesterday's transformation.  
<br> Checking transformants of yesterday with colony PCR:
<br>Mastermix (9+1 samples):
<br> 10× Taq buffer: 20μl
<br> dNTP mix 10mM: 4μl
<br> MgCl2: 12μl
<br> taq 5u/μl: 1μl
<br> Forward primer BB vector 10μM: 4μl
<br> Reverse primer BB vector 10μM: 4μl
<br> MilliQ water: 155μl
<br> PCR conditions:
<br> 1. Pre heated lid: 111 °C
<br> 2. Denaturation: 10 min. at 94°C
<br> 3. Cycle 33×:
<br>      Denaturation: 30s at 94°C
<br>      Annealing:    30s at 60°C
<br>      Extension:    2 min. at 72°C
<br> 4. Final extension: 10 min. at 72°C
<br> 5. Store infinite at 4°C
<br>
<br> Another transformation was done with the ligation mixtures of yesterday (were stored overnight in the fridge at 4°C)
<br> cI-LVA, LasR-LVA and pSB1A3-DT were digested, ligated and transformed again like yesterday, but the digestion and ligation time were increased to 1 hour.
<br> Colony PCR results: two colonies of cI-LVA seems to contain the right construct (a band of around 1000bp was seen on the 1% <br>agarose gel and this should be the size of the product with the BB vector primers).
<br> Colonies are grown overnight in LB medium with ampicillin (1μg/ml). Also the pSB1A3 vector with RBS-GFP-DT are grown <br>overnight, since there is no more plasmid available in our storage.    





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12-07-11


Very few colonies were seen on the plates of yesterday's transformation.
Checking transformants of yesterday with colony PCR:
Mastermix (9+1 samples):
10× Taq buffer: 20μl
dNTP mix 10mM: 4μl
MgCl2: 12μl
taq 5u/μl: 1μl
Forward primer BB vector 10μM: 4μl
Reverse primer BB vector 10μM: 4μl
MilliQ water: 155μl
PCR conditions:
1. Pre heated lid: 111 °C
2. Denaturation: 10 min. at 94°C
3. Cycle 33×:
Denaturation: 30s at 94°C
Annealing: 30s at 60°C
Extension: 2 min. at 72°C
4. Final extension: 10 min. at 72°C
5. Store infinite at 4°C

Another transformation was done with the ligation mixtures of yesterday (were stored overnight in the fridge at 4°C)
cI-LVA, LasR-LVA and pSB1A3-DT were digested, ligated and transformed again like yesterday, but the digestion and ligation time were increased to 1 hour.
Colony PCR results: two colonies of cI-LVA seems to contain the right construct (a band of around 1000bp was seen on the 1%
agarose gel and this should be the size of the product with the BB vector primers).
Colonies are grown overnight in LB medium with ampicillin (1μg/ml). Also the pSB1A3 vector with RBS-GFP-DT are grown
overnight, since there is no more plasmid available in our storage.