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Protocol/18
From 2010.igem.org
Protocol 18: In Vitro BioByte Assembly
Byte assembly protocol v2.0
What you will need: 1.1.5mL eppindorf tubes 2.magnet 3.Wash/binding buffer (10mM Tris 1mM EDTA pH8.0) 4.Elution buffer ? 5.5x ligase buffer 6.ligase 7.PCR cleanup kit 8.Para magnetic beads (oligo-dT25mer NEB# S1419S) 9.A18_AB anchor stock solution (0.1pM; 67ng/uL in TE) 10.AB KanR byte @ 40 ng/uL (0.06 pM/uL; gel purified in E buffer; 0.9 kbp) 11.BA Byte (0.1pM; 67ng/uL in TE)
Procedure:
Preparing AB byte Anchor: ? uL KanR AB Byte (2.2ug; 4pM) +4 uL Anchor (900 ng; 50pM) +20 uL Q-Ligase buffer (x2) +1 uL Q-ligase Total vol= 40 uL
5’ @ rm Temp followed by heat inactivation @65C for 10’
Binding rxn: mix beads with a couple of shakes folled by 10’ slow rotation Wash x2 with 50uL TE buffer + 16uL TE buffer + 4uL anc.byte (0.4pM;0.27ug total) Total vol=20uL
30’ of repeated flicking and inversion 2x Wash as above
Ligation: 6uL mQ H2O +4uL BA Byte (0.4pM;0.27ug total) 10uL(2x Q-ligase buffer) 1uL Q-ligase total vol= 20uL
5’ @ rm Temp with gentle mixing 2x Wash as above
Elution: Add 20uL of élution buffer @70C ??? Mix and remove rapidly </pre>
