Team:UCLA/Notebook/Spider Silk Genetics/Protocols/ICA Preparation
This page details solutions and reagents that should be prepared prior to beginning Iterative Capped Assembly.
Contents
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.