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