Assembly Related Protocols

From 2008.igem.org

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(New page: '''Newest EcoRI/BamHI Transfer method (7/31/08)''' Digestion: 0.5ul plasmid in entry vector 0.5ul EcoRI 0.5ul BamHI 1ul NEB2 + ATP buffer 7.5ul of water incubate the digestion for 1hr....)
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Digestion:
Digestion:
-
0.5ul  plasmid in entry vector
+
*0.5ul  plasmid in entry vector
-
0.5ul  EcoRI
+
*0.5ul  EcoRI
-
0.5ul  BamHI
+
*0.5ul  BamHI
-
1ul NEB2 + ATP buffer
+
*1ul NEB2 + ATP buffer
-
7.5ul of water
+
*7.5ul of water
-
incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.
+
*incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.
   
   
No purification step.
No purification step.
   
   
Ligation:
Ligation:
-
1ul of dilute assembly vector (or 0.5ul if undiluted)
+
*1ul of dilute assembly vector (or 0.5ul if undiluted)
-
0.5ul ligase  
+
*0.5ul ligase  
   
   
Transform. (same as usual)
Transform. (same as usual)

Revision as of 03:03, 2 August 2008

Newest EcoRI/BamHI Transfer method (7/31/08)

Digestion:

  • 0.5ul plasmid in entry vector
  • 0.5ul EcoRI
  • 0.5ul BamHI
  • 1ul NEB2 + ATP buffer
  • 7.5ul of water
  • incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.

No purification step.

Ligation:

  • 1ul of dilute assembly vector (or 0.5ul if undiluted)
  • 0.5ul ligase

Transform. (same as usual)