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

iGEM Project name 1

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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