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.

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 will pick colonies, restreak them on new plates, and do a PCR with BB primers to see whether the plasmid is 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 + KaiC_1
10	J04500 + KaiC_2
11	J04500
12	(none)