Protocol/18

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Revision as of 00:49, 26 October 2010

TEAM ALBERTA

Protocol 18: In Vitro BioByte Assembly

Byte assembly protocol v2.0


Materials & reagents:
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)
 
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

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