IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/22

From OpenWetWare

Jump to: navigation, search
iGEM Project name 1 Main project page
Previous entry      Next entry

Transfer pBAD Strong into Chloramphenicol

  • As there is a mutation in the EcoRI site on the pBAD Strong Biobrick Prefix, will transfer the insert into a Chloramphenicol backbone in order to regenerate the restriction enzyme cut site

Digest

  • Create 2 digest solutions consisting of:
    • 5uL Buffer 2
    • 29uL diH2O
    • 5uL 10X BSA
    • 0.5uL Xbal (10 units)
    • 0.5uL PstI (10 units)
    • 10ul of either pBAD strong (100ng/uL) or Chloramphenicol construction plasmid (100ng/uL)
  • Inncubate solutions for 1 hour at 37°C
  • Heat innactivate for 20 minutes at 80°C
  • To confirm the length of both the insert and the backbone, 10uL of the digest was run on a 2% agarose gel with 0.5X TBE Buffer. Both lengths were the correct size.

Ligation

  • Create a ligation solution consisting of:
    • 1.5uL digested pBAD strong
    • 1.5uL digested chloramphenicol construction plasmid
    • 2uL 10X ligation buffer
    • 12.5uL diH2O
    • 1uL ligase
  • Inncubate at 16°C for 1.5 hours
  • The ligation product was spread plated on a LB-Chlor plate and inncubated at 37°C overnight




Personal tools