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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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| - | What you will need:
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| - | 1.1.5mL eppindorf tubes
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| - | 2.magnet
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| - | 3.Wash/binding buffer (10mM Tris 1mM EDTA pH8.0)
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| - | 4.Elution buffer ?
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| - | 5.5x ligase buffer
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| - | 6.ligase
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| - | 7.PCR cleanup kit
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| - | 8.Para magnetic beads (oligo-dT25mer NEB# S1419S)
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| - | 9.A18_AB anchor stock solution (0.1pM; 67ng/uL in TE)
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| - | 10.AB KanR byte @ 40 ng/uL (0.06 pM/uL; gel purified in E buffer; 0.9 kbp)
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| - | 11.BA Byte (0.1pM; 67ng/uL in TE)
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| - |
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| - | Procedure:
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| - | Preparing AB byte Anchor:
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| - | ? uL KanR AB Byte (2.2ug; 4pM)
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| - | +4 uL Anchor (900 ng; 50pM)
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| - | +20 uL Q-Ligase buffer (x2)
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| - | +1 uL Q-ligase
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| - | Total vol= 40 uL
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| - |
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| - | 5’ @ rm Temp followed by heat inactivation @65C for 10’
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| - | Binding rxn:
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| - | mix beads with a couple of shakes folled by 10’ slow rotation
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| - | Wash x2 with 50uL TE buffer
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| - | + 16uL TE buffer
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| - | + 4uL anc.byte (0.4pM;0.27ug total)
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| - | Total vol=20uL
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| - |
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| - | 30’ of repeated flicking and inversion
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| - | 2x Wash as above
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| - | Ligation:
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| - | 6uL mQ H2O
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| - | +4uL BA Byte (0.4pM;0.27ug total)
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| - | 10uL(2x Q-ligase buffer)
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| - | 1uL Q-ligase
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| - | total vol= 20uL
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| - | 5’ @ rm Temp 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 @70C ???
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| - | Mix and remove rapidly
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| - | </pre>
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| - | [[Team:Alberta/Notebook/protocols| Back]]
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| - | {{Team:Alberta/endMainContent}}
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