29/07/13

From 2013.igem.org

(Difference between revisions)
(Ran the gel from the 26/07/2013 again for 45min)
Line 30: Line 30:
*Add the following substances  
*Add the following substances  
**11.1ul of dH2O
**11.1ul of dH2O
-
**1ul of QS ligase
+
**1ul of QS Ligase *
-
**5ul of 4xQS buffer (vortex before use)
+
**5ul of 4xQS Buffer (vortex before use)
-
**mix thoroughly by pipetting  
+
**Mix thoroughly by pipetting  
-
*incubate at room temperature for 5 min to create cohesive ends
+
*Incubate at room temperature for 5 min to create cohesive ends
-
*run 2.5-5ul of the ligation mixture onto an agarose gel to check ligation efficiency
+
*Run 2.5-5ul of the ligation mixture onto an agarose gel to check ligation efficiency
-
*transform it into competent cells (E.coli)
+
*Transform it into competent cells (E.coli)
 +
 
 +
*For this experiment, two separate samples will be run; a control and the actual ligation. For the control, no QS Ligase is added but its equivalent volume (1ul) is made up by dH2O.

Revision as of 16:10, 29 July 2013

Contents

Ran the gel from the 26/07/2013 again for 45min

IGEM lim restr dig 2907130 2ndrun0.1.jpg

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

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

Ligation of insert to vector

  • Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
  • Add the following substances
    • 11.1ul of dH2O
    • 1ul of QS Ligase *
    • 5ul of 4xQS Buffer (vortex before use)
    • Mix thoroughly by pipetting
  • Incubate at room temperature for 5 min to create cohesive ends
  • Run 2.5-5ul of the ligation mixture onto an agarose gel to check ligation efficiency
  • Transform it into competent cells (E.coli)
*For this experiment, two separate samples will be run; a control and the actual ligation. For the control, no QS Ligase is added but its equivalent volume (1ul) is made up by dH2O.