IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16
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To-do
- Western blots
Inoculate several cultures of GFP dev. for Western blotting this afternoon- Take regular measurements of culture OD
- Western blot
- Construct creation
- Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there
- Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday
- Digest a large amount of KaiA/B/C\X-P
- Gel-purify
- RBS + KaiC construct
- Miniprep the B0034 cultures
- Digest B0034\S-P
- Ligate KaiC\X-P + B0034\S-P
- Transform KaiC\X-P + B0034\S-P
Inoculation R0010 + E0241
10 inoculations of the R0010 + E0241 were made:
- 10 mL LB amp & 1 ul R0010 + E0241
- 10 mL LB amp & 1 ul R0010 + E0241 (IPTG - not yet added)
- 10 mL LB amp & 8 ul R0010 + E0241
- 10 mL LB amp & 8 ul R0010 + E0241 (IPTG - not yet added)
- 10 mL LB amp & 64 ul R0010 + E0241
- 10 mL LB amp & 64 ul R0010 + E0241 (IPTG - not yet added)
- 10 mL LB amp & 256 ul R0010 + E0241
- 10 mL LB amp & 256 ul R0010 + E0241 (IPTG - not yet added)
- 10 mL LB amp & 4.1 mL R0010 + E0241
- 10 mL LB amp & 4.1 mL R0010 + E0241 (IPTG - not yet added)
Midiprep of KaiABC
Ligation test and replating
Each reaction:
- 8 µL PCR Supermix
- 1 µL VF2 primers @ 2uM
- 1 µL VR primers @2uM
- colony or 1 uL template or nothing
18 RXNS:
- 8*20=160uL supermix
- 1*20 = 20uL VF2
- 1*20 = 20uL VR
PCR schedule:
- 95C for 15'00
- Do 30 times:
- 95C for 0'30
- 55C for 0'30
- 72C for 2'00
- 72C for 10'00
- 4C forever
