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Team:Alberta/Notebook/protocols/invitro biobyte assembly
In Vitro BioBytes Assembly 2.0
Reagents:
- 1.5mL Eppendorf 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:
- Add in a reaction:
| 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 anchor 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 elution buffer @70oC.
- Mix and remove rapidly.
