IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-13

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Gel purification, Ligation and transformation of GeneArt Plasmids, natural KaiA into J04500 backbone

I gel purified the inserts pre-ligation, but the nanodrop gave extremely low (eg. negative) numbers. Then, I used an absolute ratio of 1:3 vector-insert instead of the normal molar ratio. DO NOT USE THIS PROTOCOL FOR SUBSEQUENT LIGATIONS!

Ligation for samples the nanodrop hates

  • BB vector J04500 \SP: 2.5uL
  • BB insert KaiA, B, or C \XP: 7.5uL

Protocol:

  • Mix above ratios + 10uL #1 + 1uL #3
    • BE sure to mix #1 (ligation buffer) well especially!
  • Sit at bench for ~30min
  • Put back on ice

Transformation

For the transformation I did 5 samples:

  • 1 exp. (50uL) natural KaiA
  • 1 exp. (50uL) synthetic KaiA
  • 1 exp. (50uL) synthetic KaiB
  • 1 exp. (50uL) synthetic KaiC
  • 1 (25uL) positive
  • 1 (25uL) negative

Ice incubation 30m, 30s@42C, 2m@ice, SOC@1h@37C. Plated on LBCarb. Should be ready around 6PM to see.

Liquid cultures of KaiA/B/C in Top10, J04450 + pSB4A3 in Top10 and Top10F'

Growth

  • KaiA
  • KaiB
  • KaiC
  • F2-3
  • F2-4

No growth

  • 101
  • 102
  • F1
  • F2-1
  • F2-2

I made frozen stocks of all cultures. 600 uL culture + 400 uL 50% glycerol.

Liquid culture of J04500

The large culture of J04500 that we inoculated yesterday for midiprepping appears to be red. We think we accidentally inoculated J04450 instead of J04500.

Miniprep of KaiA/B/C + GeneART plasmid in Top10

Eluted in 50 uL H2O.

Restreak and PCR of J04500 + KaiA/B/C transformants

The plates that Peng streaked last night (see beginning of page) showed growth by around 8PM. We picked colonies, restreaked them on new plates, and did a PCR with BB primers to see whether the ligation and transformation of KaiA/B/C into J04500 was correct.

Reaction # DNA
1 J04500 + KaiAg_1
2 J04500 + KaiAg_2
3 J04500 + KaiAs_1
4 J04500 + KaiAs_2
5 J04500 + KaiAs_3
6 J04500 + KaiAs_4
7 J04500 + KaiB_1
8 J04500 + KaiB_2
9 J04500 + KaiB_3
10 J04500 + KaiC_1
11 J04500 + KaiC_2
12 J04500
13 (none)

Each reaction:

  • 8 µL PCR Supermix
  • 1 µL VF2 primers
  • 1 µ VR primers
  • colony or 1 uL template or nothing

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