▪ Aug 2
esa Quorum Sensing
☀ Adrian ☀
Recreating backbone
| pSB1A2 EcoRI/PstI |
| H2O | 22ul |
| pSB1A2-GFP | 10ul |
| Buffer | 4ul |
| EcoRI | 2ul |
| PstI | 2ul |
| Total | 40ul |
Anaerobic Promoter
☀ Adrian ☀
Inoculated colony 3,7 of FNR GFp selected positive clones for miniprep
|
▪ Aug 3
Anaerobic Promoter
☀ Yi Han ☀
Miniprep of colony 3, 7 and sent for sequencing with GFP-R and FP_VF2 -> results show there is GFP but no FNR.
esa Quorum Sensing
☀ Adrian ☀
| Fusion PCR for workshop |
| H2O | 111ul |
| Buffer | 40ul |
| MgCl2 | 16ul |
| dNTP | 16ul |
| OFP_esaRBS_GFP_ver2 | 8ul |
| ORP_XhoI_retarded | 8ul |
| GoTaq | 1ul |
| Fragment 1 | 1ul |
| Fragment 2 | 1ul |
95ºC (3min) -> 95ºC (30s) -> 60ºC (30s) -> 72ºC (1.5min) -> 72ºC (10min)
for 25 cycles
| EsaR Master Plate Test |
| Colony PCR |
| H2O | 222ul |
| Buffer | 80ul |
| MgCl2 | 32ul |
| dNTP | 32ul |
| VF2 | 16ul |
| VR | 16ul |
| GoTaq | 2ul |
| Total | 400ul |
For 10 colonies
|
▪ Aug 6
Anaerobic Promoter
☀ Yi Han ☀
Transform pNirB+gfp from the Biobricks Registry into BL21 and dH5alpha
|
▪ Aug 8
Anaerobic Promoter
☀ Yi Han ☀
8 minipreps of 4 clones from BL21 pNirB+gfp and 4 clones from dH5alpha pNirB+gfp
| RE digest |
| EcoRI | 9ul |
| PstI | 9ul |
| Cutsmart Buffer | 45ul |
| DNA of 1ug | 6.7ul |
| H2O | 36.3ul |
▶ Result after running gel: All clones are positive for pNirB+gfp
|
