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

Contents 1 To-do 2 Miniprep of B0034 3 Inoculation R0010 + E0241 4 Midiprep of KaiABC 5 Ligation test and replating 6 Innoculation of GFP Device from Nick's Plate 7 Digest of Kai\X-P and B0034\S-P

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

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.