Team:UCLA/Notebook/Spider Silk Genetics/Protocols/ICA Preparation

iGEM UCLA




This page details solutions and reagents that should be prepared prior to beginning Iterative Capped Assembly.

Solutions

2x Binding and Wash (BW) Buffer

  • 2 M NaCl
  • 10 mM Tris-Cl, pH 8.0
  • 1 mM ethylenediaminetetracetic acid (EDTA), pH 8.0
  • 0.2% v/v Tween-20
  • For 50 mL of 2x BW Buffer:
    • 5.844 g NaCl
    • 500 uL of 1 M Tris-Cl, pH 8.0
    • 100 uL of 0.5 M EDTA, pH 8.0
    • 100 uL Tween-20
    • Add ddH2O to 50 mL.

0.5x BW Buffer

  • Make a 1:4 dilution of 2x BW buffer in ddH2O
  • For 10 mL of 0.5x BW Buffer.
    • Dilute 2.5 mL of 2x BW Buffer to 10 mL.

ICA Elution Buffer

  • 0.01% v/v Tween-20 in ddH2O
  • For 10 mL of Elution Buffer
    • 1 uL Tween in 10 mL of ddH2O.

Initiator, Terminator, and Capping Oligo Preparation

  • Initiator and Terminator
    • Mix equal volumes of the two relevant ssDNA oligos at 100 uM each.
    • Heat at 95 C and ramp down to 25 C at 0.1 C/s.
    • After Heat treatment, prepare a 50 nM solution of the Initiator, and a 5 uM solution of the Terminator
  • Caps
    • Dilute Caps to 5 uM.
    • Heat at 95 C and ramp down to 25 C at 0.1 C/s.