29/07/13

From 2013.igem.org

(Difference between revisions)
Line 3: Line 3:
*Three different reactions  
*Three different reactions  
*Master Mix (changes were made from the protocol)
*Master Mix (changes were made from the protocol)
-
**5ul NEB Buffer 2
+
**5ul NEB Buffer 3.1
**0.5ul of EcoRI
**0.5ul of EcoRI
**0.5ul of PstI
**0.5ul of PstI
Line 10: Line 10:
**4ul of linearized backbone
**4ul of linearized backbone
**4ul of enzyme master mix
**4ul of enzyme master mix
 +
*digest at 37C for 30min
 +
*heat kill at 80C for 20min
 +
#Digestion of the limonene biobrick plasmid backbone (BBa_K118025)
 +
*Using clone 1.1 purified plasmid DNA
 +
*For the reaction adding:
 +
**5ul NEB Buffer 3.1
 +
**2ul of EcoRI
 +
**2ul of PstI
 +
**2.5ul of dH2O
 +
**38.5ul of DNA
*digest at 37C for 30min
*digest at 37C for 30min
*heat kill at 80C for 20min
*heat kill at 80C for 20min

Revision as of 14:10, 29 July 2013

  1. Ran the gel from the 26/07/2013 again for 45min
  2. Digestion of plasmid backbone (pSB1C3)
  • Three different reactions
  • Master Mix (changes were made from the protocol)
    • 5ul NEB Buffer 3.1
    • 0.5ul of EcoRI
    • 0.5ul of PstI
    • 19ul of dH2O
  • For each reaction add:
    • 4ul of linearized backbone
    • 4ul of enzyme master mix
  • digest at 37C for 30min
  • heat kill at 80C for 20min
  1. Digestion of the limonene biobrick plasmid backbone (BBa_K118025)
  • Using clone 1.1 purified plasmid DNA
  • For the reaction adding:
    • 5ul NEB Buffer 3.1
    • 2ul of EcoRI
    • 2ul of PstI
    • 2.5ul of dH2O
    • 38.5ul of DNA
  • digest at 37C for 30min
  • heat kill at 80C for 20min