IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-16

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== Innoculation of GFP Device from Nick's Plate ==
== Innoculation of GFP Device from Nick's Plate ==

Revision as of 08:18, 17 August 2006



Contents

To-do

  • Western blots
    • Inoculate several cultures of GFP dev. for Western blotting this afternoon
    • Take regular measurements of culture OD
    • Western blot (delayed until tomorrow because we don't seem to have GFP)
  • 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
      • Gel-purify
      • Ligate KaiC\X-P + B0034\S-P
      • Transform KaiC\X-P + B0034\S-P

Miniprep of B0034

Title says it all; 2 60uL samples of B0034 in the freezer, and glycerol stock.

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

We had 18 colonies grow up; see the image at the right. For each of these, I plated and did a colony PCR to test for the insert. For the first 8, I also innoculated, though it may not work.

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


Results:

I'm confused.... the + control is B0034 (the RBS), for reference; the actual coding sequence is i think 33bp or something really short, so basically add on the size of the band in the gel to the expected KaiA,B,and C sizes.

EDIT: Actually, the previous ligation was not a self-ligation like thought. Look at http://www.openwetware.org/wiki/Image:2006_08_14_egel2.jpg, we expect a 550 bp band (like + control) if it self-ligated but it actually is a 300bp band...

EXPECTED

  • KaiA from VF to VR2: 855bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 1.4kb
  • KaiB: 309bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 859bp
  • KaiC: 1560bp + 536bp (VF2-VR on J04500) - 6bp+20bp (prefixes) = 2110bp
  • B0034 Positive: 250bp (VF2-VR) = 250bp
  • Nothing: 536bp
click for legend
click for legend
click for legend
click for legend


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 GAX, P37C16 hrs
KaiB in GAX, P37C16 hrs
KaiC in GAX, P37C16 hrs
B0034 in pSB1A2S, P37C16 hrs
KaiA in GAX, P45C16 hrs
KaiB in GAX, P45C16 hrs
KaiC in GAX, P45C16 hrs
B0034 in pSB1A2S, P45C16 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.
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