IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-9

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Short-term plan

  • J04500 (Lac promoter + RBS in an AmpR and KanR backbone)
    • Inoculate the J04500 we transformed yesterday
    • Make frozen stocks of the liquid culture
    • Miniprep the culture
    • Digest the plasmids with S-P
    • Gel-purify
  • KaiA
    • We already have high-copy plasmids containing KaiA from Peng's massive miniprep on Monday
    • Select the DNA that showed promise in the X-P digest gel, and digest it with X-P again for a longer time
    • Gel-purify the KaiA\X-P, and maybe run it on a gel with undigested KaiA for comparison
  • Ligate the J04500\S-P with KaiA\X-P to make a BB with KaiA after a Lac promoter and RBS

Misplated (?) J04500 in Top10F'

Yesterday's J04500 in Top10F' transformants appear to be misplated. The negative control has growth, while one experimental plate has growth and the other is clear. I think I switched one of the experimental plates with the negative control when spreading the transformant cells. It seems unlikely that one of the experimental plates would be clear and the other would show growth if contamination were the problem.

Inoculation of J04500 transformants

We will pick a colony from the experimental plate that showed growth, and inoculate it in LB-Amp. The J04500 plasmid has both Amp and Kan resistance. Therefore it's unlikely that our colony from the LB-Kan plate will grow in LB-Amp unless it's our plasmid.

Retransformation of J04500

Just in case we really did have contamination in yesterday's transformation, we redid the transformation (with Top10 cells instead of Top10F', because we should conserve the Top10F' cells until we need tight Lac repression).

XP digestion of possibly working ligation

KaiA+bkb 2,4, and 7 showed possibilty of a working liation.

  • 19.25 uL DNA (~15 ng/uL gives 0.288ug DNA)
  • 2 uL H2O
  • 2.5 uL buffer (EcoRI buffer or Buffer 3)
  • 0.5 uL enzyme 1 (XbaI)
  • 0.5 uL enzyme 2 (PstI)
  • 0.25 uL BSA

25 uL total volume.

MM:

  • 4x2= 8 dh20
  • 4x2.5= 10 buffer 3
  • 4x0.5 = 2 XbaI
  • 4x0.5 = 2 PstI
  • 4x0.25 = 1 BSA

pipet 5.75 into each.

then load rest of samples and run on gel side-by-side.

Inoculation of BBa_J04450 & pSB4A3

The transformation of the RFP from BBa_J04450 and the pSB4A3 backbone was plated two days ago, and this morning, the plates showed some growth. The cultures were inoculated in 2 mL of LB and 2 uL of 50mg/mL amp. One additional note: the colonies that were extracted seemed dry, so the inoculations may not have worked out.

More Inoculations

An inoculation of the GFP device (R0010 & E0241) was inoculated in 8 mL of LB amp.

There was an additional inoculation of J04500 in Top10F' in 8 mL of LB amp.

Digestion Assay for 8/10

Label DNA Amount Water Enzymes Amount NEB buffer BSA (100x) Total
Digests 1 R0010 & E0241 19.25 µL 2 µL XbaI & PstI 0.5 µL, 0.5 µL Buffer 3 (2.5 µL) 0.25 µL 25 µL
2 pSB4A3 bkb 19.25 µL 2 µL SpeI & PstI 0.5 µL, 0.5 µL Buffer 2 (2.5 µL) 0.25 µL 25 µL
3 R0010 & E0241 19.25 µL 1 µL XbaI & PstI 1 µL, 1 µL Buffer 3 (2.5 µL) 0.25 µL 25 µL
4 pSB4A3 bkb 19.25 µL 1 µL SpeI & PstI 1 µL, 1 µL Buffer 2 (2.5 µL) 0.25 µL 25 µL
Master Mixes 1 R0010 & E0241 115.5 µL 12 µL XbaI & PstI 3 µL, 3 µL Buffer 3 (15 µL) 1.5 µL 150 µL
2 pSB4A3 bkb 115.5 µL 12 µL SpeI & PstI 3 µL, 3 µL Buffer 2 (15 µL) 1.5 µL 150 µL
3 R0010 & E0241 115.5 µL 6 µL XbaI & PstI 6 µL, 6 µL Buffer 3 (15 µL) 1.5 µL 150 µL
4 pSB4A3 bkb 115.5 µL 6 µL SpeI & PstI 6 µL, 6 µL Buffer 2 (15 µL) 1.5 µL 150 µL

We will be digesting these reactions for 2, 4, 6, 12, & 16 hours

The 2, 4, 6, and 12 hour digestions will be done manually (both the incubation and the heat shock), and then placed in a freezer until all the digestions are complete.

The 16 hour digestion will utilize a PCR machine; also, heat shocking of 20 minutes will succeed the digestion.