Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/11

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February 9, 2013

Golden Gate Assembly

  • Mediated assembly
    • The volume of purified DNA (x) needed to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
    • Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20
DNA length (bp) ng/μL vol. dH2O
H2B 410 77.995 1.4 18.6
LOV 461 80.443 1.5 18.5
pSB1A3 2000 77.4 6.7 13.3


Reagent Assembly Control
20 fmol H2B 1.0 0
20 fmol LOV 1.0 0
20 fmol pSB1A3 1.0 1.0
10x T4 ligase buffer (Promega) 1.0 1.0
T4 ligase (NEB) 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
Total vol. 10.0 10.0
  • Bacterial transformation
    • Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
    • Incubate on ice for 2 min., heat shock at 42°C for exactly 45 sec., immediately place on ice.
    • Add 800 μL sterile SOC medium.
    • Grow with shaking at 37°C for 30 min.
    • Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp.
    • Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic.
    • Plate cells on pre-warmed LB agar + antibiotic. Grow overnight at 37°C. Quick-transormation (e.g., DH5α-Turbo) is not recommended



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