IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-23

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Ligation test

With new J04500 /SP and new KaiC /XP, attempting ligation again; this time following up on the advice Roche gave me about using highly concentrated DNA and 2uL of DNA dilution buffer.

The NEB Kit says not to go above 10ng/uL rxn, or otherwise linear DNA forms... but for KaiC I'm going to try it because lower concentrations have consistently not worked - I'll stick to 300ng total sample, or 150% overestimation.

  • J04500 /SP + KaiC /XP concentrated from 8/21.
    • BB vector: (110.6ng @ 110.6 ng/uL) = 1 uL
    • BB insert: 3*(~2100/3400)*110.6 = 204.9 ng @ (60 ng/uL) = 3.42 uL
    • 1x DNA Dilution buffer: 2uL
    • ddH20: 3.58uL
  • J04500 /SP + KaiA /SP from 8/22 (Labeled C2)
    • BB vector: 110.6ng @ (110.6 ng/uL) = 1 uL
    • BB insert: 3*(~2100/3400)*110.6 = 204.9 ng @ (24.2ng/uL) = 8.47 uL Scaled to 7uL
    • 1x DNA Dilution buffer: 2uL
  • J04500 /SP + KaiA /SP from 8/22 (Labeled C1), concentrated down to ~5uL from 30uL
    • BB vector: 330ng @ (110.6 ng/uL) = 3 uL
    • BB insert: 8uL of C1 DNA, around 60ng/uL = 480ng.
    • 1x DNA Dilution buffer: 2uL

Let the ligation sit 30 min. Then, using 3 vials of Top10F:

  • 'c2' 19uL ligation product 30uL cells
  • 'c2' 2uL ligation product 30uL cells
  • 'c1 conc.' 21uL ligation product 30uL cells
  • 'old' 19uL ligation product 30uL cells
  • 'old' 2uL ligation product 30uL cells
  • 'positive' The J04500 plasmid Hetmann miniprepped @21uL, 15uL of cells
  • 'negative' 21uL water ,15uL cells.

30 min ice, 42C@30s heat, 2min ice, ~80min SOC, plating all on KAN except for the positive control, which is on CARB. Ready around 4pm.

Transformant plates

As of 12:30AM on 2006-8-24, there are still no colonies on the plates. I inoculated 4 cultures of the positive control we used (J04500 from Hetmann's midiprep), in 5 mL LB Amp, with 5 uL IPTG added.
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