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.
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