IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16: Difference between revisions
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'''B0034\S-P master mix''' | '''B0034\S-P master mix''' | ||
* | * 10 µL NEBuffer 2 | ||
* | * 2 µL SpeI | ||
* | * 2 µL PstI | ||
* | * 1 µL 100x BSA | ||
Pipette 3.75 µL into each reaction. | Pipette 3.75 µL into each reaction. |
Revision as of 17:50, 16 August 2006
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To-do
- Western blots
Inoculate several cultures of GFP dev. for Western blotting this afternoonTake 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 thereMidiprep 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
The KaiABC genes were eluted (redissolved) in 200 mL of H2O.
- KaiA: 279.1 ng/uL
- KaiB: 285.2 ng/uL
- KaiC: 409.7 ng/uL + 409.5 ng/uL
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
19 RXNS: (18 + 1 pos, which is B0034)
- 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
Innoculation of GFP Device from Nick's Plate
4 Innoculations were made of GFP (R0010 + E0241) from Nick's bright green plate. The cells could be dead, though, so no guarantees. All 4 innoculations were made in 10mL LB + Amp. Also made a negative control of 10mL LB + Amp.
Digest of Kai\X-P and B0034\S-P
Plasmid | Enzymes | Incubation temperature | Incubation time |
KaiA in GA | X, P | 37C | 16 hrs |
KaiB in GA | X, P | 37C | 16 hrs |
KaiC in GA | X, P | 37C | 16 hrs |
B0034 in pSB1A2 | S, P | 37C | 16 hrs |
KaiA in GA | X, P | 45C | 16 hrs |
KaiB in GA | X, P | 45C | 16 hrs |
KaiC in GA | X, P | 45C | 16 hrs |
B0034 in pSB1A2 | S, P | 45C | 16 hrs |
Kai\X-P reactions
- 8 µL midiprepped DNA
- 2.5 µL NEBuffer 3
- 0.5 µL XbaI
- 0.5 µL PstI
- 0.25 µL 100x BSA
- 13.25 µL H2O
Kai\X-P master mix
- 17.5 µL NEBuffer 3
- 3.5 µL XbaI
- 3.5 µL PstI
- 1.75 µL 100x BSA
- 92.75 µL H2O
Pipette 17 µL into each reaction.
B0034\S-P reactions
- 21.25 µL DNA
- 2.5 µL NEBuffer 2
- 0.5 µl SpeI
- 0.5 µL PstI
- 0.25 µL 100x BSA
B0034\S-P master mix
- 10 µL NEBuffer 2
- 2 µL SpeI
- 2 µL PstI
- 1 µL 100x BSA
Pipette 3.75 µL into each reaction.