IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16: Difference between revisions

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** Western blot
** Western blot
* Construct creation
* Construct creation
** Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there
** <s>Restreak and do colony PCRs of the Kai\X-P + J04500\S-P transformants to check if our insert is there</s>
** <s>Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday</s>
** <s>Midiprep the KaiA/B/C in GA plasmid that we grew up from frozen stocks yesterday</s>
*** Digest a large amount of KaiA/B/C\X-P
*** Digest a large amount of KaiA/B/C\X-P
*** Gel-purify
*** Gel-purify
** RBS + KaiC construct
** RBS + KaiC construct
*** Miniprep the B0034 cultures
*** <s>Miniprep the B0034 cultures</s>
*** Digest B0034\S-P
*** Digest B0034\S-P
*** Ligate KaiC\X-P + B0034\S-P
*** Ligate KaiC\X-P + B0034\S-P
Line 74: Line 74:
== Innoculation of GFP Device from Nick's Plate ==
== 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.
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 ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Plasmid'''
| align="center" style="background:#f0f0f0;"|'''Enzymes'''
| align="center" style="background:#f0f0f0;"|'''Incubation temperature'''
| align="center" style="background:#f0f0f0;"|'''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 uL midiprepped DNA
*

Revision as of 17:41, 16 August 2006

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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

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 uL midiprepped DNA
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