User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/25

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October 25, 2010
1. Extract plasmid from colonies 1-10 incubated on October 24, the plasmid should contain: pSB3K3 + J23101 + ΔRBS + GFP E004
 * Plamid extraction was made using the QIAprep Spin Miniprep Kit.

2. Made PCR to test pSB3K3 + J23101 + ΔRBS + GFP E004 construction.
 * PCR will be done with Platinum Taq Polymerase.
 * Primers used: Preffix FWD-Suffix REV.
 * Template for the reaction is extracted plasmid from the transformation with this construction (Colonies 1-10, tubes are marked the same way).
 * Positive control: BBa_I51020 (p19).


 * PCR with Platinum Taq DNA polymerase -> Volume (ul)
 * 10X PCR Buffer minus M -> 5
 * 10mM dNTP mixture -> 1
 * 50mM MgCl2 -> 2.5
 * Primer mix (10uM each) -> 2
 * Platinum Taq DNA Pol -> 0.4
 * Template DNA -> 1
 * HPLC -> 38.1
 * Total volume -> 50


 * If primers are separated and in concentration 5uM, use 1ul of each one.


 * Thermocycler program:
 * 1. 95ºC 5 min
 * 2. 35 cycles
 * -95ºC 45 seg
 * -55ºC 45 seg
 * -72ºC 1:00 min
 * 3. 72ºC 10 min
 * 4. Hold 4ºC

3. Run gel to verify PCR’s made on October 23 and 25.



￼Lanes: 1) Green ladder; 2) MinBP + ΔRBS + GFP E004 Colony 1 (C1) (expressing RFP); 3) MinBP + ΔRBS + GFP E004 (C7); 4) BBa_I51020 Ctrl + 23 Oct.; 5-14) pSB3K3 + J23101 + ΔRBS + GFP E004 (Colonies 1-10, respectively); 15) BBa_I51020 Ctrl + 25 Oct.

4. Incubate overnight in M9 minimum medium suplmented with glycerol (0.4%) the following constructions:
 * MinBP + ΔRBS + GFP E004 in pSB1C3 (37º and 25º C, in the dark, colonies 7 & X)
 * J23101 + ΔRBS + GFP E004 in pSB3K3 (37ºC, colonies 3 & 5)
 * MinBP in pSB4A5 (37ºC and 25ºC, in the dark, colonies 3 & 5)


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