Team:Alberta/Notebook/protocols/invitro biobyte assembly

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Revision as of 17:12, 26 October 2010

TEAM ALBERTA

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:

  • 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 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.


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