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.
- 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)