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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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


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