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

  8/22 Entry  8/23 Entry  8/25 Entry  

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.