Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/Protocols/ICA Preparation"
Vinsonclam (Talk | contribs) (→ICA Elution Buffer) |
Vinsonclam (Talk | contribs) (→Initiator, Terminator, and Capping Oligo Preparation) |
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**Mix equal volumes of the two relevant ssDNA oligos at 100 uM each. | **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. | **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 | *Caps | ||
**Dilute Caps to 5 uM. | **Dilute Caps to 5 uM. | ||
**Heat at 95 C and ramp down to 25 C at 0.1 C/s. | **Heat at 95 C and ramp down to 25 C at 0.1 C/s. | ||
Latest revision as of 16:22, 25 August 2015
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.