Team:Cambridge/Protocols/Gibson Assembly

From 2011.igem.org

(Difference between revisions)
m (→Gibson Assembly)
(→Practice)
Line 8: Line 8:
===Practice===
===Practice===
 +
Master Mix for Gibson Assembly
 +
{| border="1px"
 +
 +
!Reagent
 +
!Volume/µl
 +
|-
 +
|Taq ligase (40u/µl)
 +
|50
 +
|-
 +
|5x isothermal buffer
 +
|100
 +
|-
 +
|T5 exonuclease (1u/µl)
 +
|2
 +
|-
 +
|Phusion polymerase (2u/µl)
 +
|6.25
 +
|-
 +
|Nuclease-free water
 +
|-216.75
 +
|Total
 +
|375
 +
|-
 +
|}
 +
 +
Master Mix is 1.33x concentrated
 +
 +
In order to join 3 fragments together add 1µl of each fragment (previously amplified by PCR) to a PCR tube along with 9µl of the master mix above.
===Safety===
===Safety===
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Revision as of 15:08, 12 July 2011

Loading...
OVERVIEW
home

Gibson Assembly

Theory

Practice

Master Mix for Gibson Assembly

Reagent Volume/µl
Taq ligase (40u/µl) 50
5x isothermal buffer 100
T5 exonuclease (1u/µl) 2
Phusion polymerase (2u/µl) 6.25
Nuclease-free water
Total 375

Master Mix is 1.33x concentrated

In order to join 3 fragments together add 1µl of each fragment (previously amplified by PCR) to a PCR tube along with 9µl of the master mix above.

Safety


Retrieved from "http://2011.igem.org/Team:Cambridge/Protocols/Gibson_Assembly"