"
Protocol/18
From 2010.igem.org
(Difference between revisions)
| Line 12: | Line 12: | ||
Protocol 18: In Vitro BioByte Assembly | Protocol 18: In Vitro BioByte Assembly | ||
| + | |||
Byte assembly protocol v2.0 | Byte assembly protocol v2.0 | ||
| - | + | ||
| - | + | Reagents: | |
| - | + | ||
| - | + | * 1.5mL eppindorf tubes | |
| - | + | * Magnet | |
| - | + | * Wash/binding buffer (10mM Tris 1mM EDTA pH8.0) | |
| - | + | * Elution buffer ? | |
| - | + | * 5x ligase buffer | |
| - | + | * Ligase | |
| - | + | * PCR cleanup kit | |
| - | + | * Para magnetic beads (oligo-dT25mer NEB# S1419S) | |
| - | + | * A18_AB anchor stock solution (0.1pM; 67ng/uL in TE) | |
| + | * AB KanR byte @ 40 ng/uL (0.06 pM/uL; gel purified in E buffer; 0.9 kbp) | ||
| + | * BA Byte (0.1pM; 67ng/uL in TE) | ||
| + | |||
Procedure: | Procedure: | ||
| - | Preparing AB byte Anchor: | + | *Preparing AB byte Anchor: |
| - | + | {| | |
| - | + | |KanR AB Byte (2.2ug; 4pM) || 5uL | |
| - | + | |- | |
| - | + | |Anchor (900 ng; 50pM) || 4uL | |
| - | Total | + | |- |
| + | |Q-Ligase buffer (x2) || 20uL | ||
| + | |- | ||
| + | |Q-ligase || 1uL | ||
| + | |- | ||
| + | |Total || 40uL | ||
| + | |} | ||
| + | *5 minutes @ R/T followed by heat inactivation @65<sup>o</sup>C for 10 minutes. | ||
| - | |||
| - | Binding | + | Binding: |
| - | + | * Mix beads with a couple of shakes followed by 10 minutes slow rotation. | |
| - | Wash x2 with 50uL TE buffer | + | * Wash x2 with 50uL TE buffer |
| - | + | * Add anc.byte ((0.4pM;0.27ug) and top to 20uL with TE. | |
| - | + | * 30 minutes of repeated flicking and inversion | |
| - | + | * 2x Wash as above | |
| - | |||
| - | |||
Ligation: | Ligation: | ||
| - | 6uL | + | * Add: |
| - | + | {| | |
| - | + | |MilliQ water || 6uL | |
| - | + | |- | |
| - | + | |BA Byte (0.4pM;0.27ug total) || 4uL | |
| + | |- | ||
| + | |2x Q-ligase buffer || 10uL | ||
| + | |- | ||
| + | |Q-ligase || 1uL | ||
| + | |- | ||
| + | |Total || 20uL | ||
| + | |} | ||
| + | * 5 minutes @ R/T with gentle mixing. | ||
| + | * 2x Wash as above | ||
| - | |||
| - | |||
Elution: | Elution: | ||
| - | Add 20uL of élution buffer @ | + | * Add 20uL of élution buffer @70<sup>o</sup>C. |
| - | Mix and remove rapidly | + | * Mix and remove rapidly. |
| - | + | ||
[[Team:Alberta/Notebook/protocols| Back]] | [[Team:Alberta/Notebook/protocols| Back]] | ||
{{Team:Alberta/endMainContent}} | {{Team:Alberta/endMainContent}} | ||
Revision as of 02:33, 26 October 2010
Protocol 18: In Vitro BioByte Assembly
Byte assembly protocol v2.0
Reagents:
- 1.5mL eppindorf tubes
- Magnet
- Wash/binding buffer (10mM Tris 1mM EDTA pH8.0)
- Elution buffer ?
- 5x ligase buffer
- Ligase
- PCR cleanup kit
- Para magnetic beads (oligo-dT25mer NEB# S1419S)
- A18_AB anchor stock solution (0.1pM; 67ng/uL in TE)
- AB KanR byte @ 40 ng/uL (0.06 pM/uL; gel purified in E buffer; 0.9 kbp)
- BA Byte (0.1pM; 67ng/uL in TE)
Procedure:
- Preparing AB byte Anchor:
| KanR AB Byte (2.2ug; 4pM) | 5uL |
| Anchor (900 ng; 50pM) | 4uL |
| Q-Ligase buffer (x2) | 20uL |
| Q-ligase | 1uL |
| Total | 40uL |
- 5 minutes @ R/T followed by heat inactivation @65oC for 10 minutes.
Binding:
- Mix beads with a couple of shakes followed by 10 minutes slow rotation.
- Wash x2 with 50uL TE buffer
- Add anc.byte ((0.4pM;0.27ug) and top to 20uL with TE.
- 30 minutes of repeated flicking and inversion
- 2x Wash as above
Ligation:
- Add:
| MilliQ water | 6uL |
| BA Byte (0.4pM;0.27ug total) | 4uL |
| 2x Q-ligase buffer | 10uL |
| Q-ligase | 1uL |
| Total | 20uL |
- 5 minutes @ R/T with gentle mixing.
- 2x Wash as above
Elution:
- Add 20uL of élution buffer @70oC.
- Mix and remove rapidly.
