October 15th, 2010
1. Test cells in the microscope for GFP expression.
- pSB4A5 + MinBP + ΔRBS + GFP E004
- I20269 (pSB3K3 + J23101 + GFP E0040)
2. Make PCR to test ligation pSB4A5 + MinBP + ΔRBS + GFP E004
- 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 1-3, 6-10, tubes are marked the same way).
- Positive control: BBa_I51020.
- 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
- Number of samples -> 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 1:10 min
- 3. 72ºC 10 min
- 4. Hold 4ºC
3. Make the following ligation:
- pSB3K3 + J23101 SpeI-PstI (3ul) with ΔRBS + GFP E004 XbaI-PstI (5ul)
- Ligation methods (Total volume 20ul):
- DNA -> Volume of each part to be ligated is indicated above.
- Buffer for T4 DNA ligase 5X -> 4ul (Final concentration 20%)
- T4 DNA ligase -> 1ul
- HPLC -> Complete total volume (20ul)
- Incubate overnight at 16ºC
- Mix well (vortex) buffer and reaction tubes, unfreeze buffer on ice.