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

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

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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.