Team:HKU-Hong Kong/Lab Diaries

Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm)

1. tetO2 -1 (DNA binding site)
   1. Reverse PCR was used to insert tetO2 -1 into pEGFP-loxp-km-loxp (template DNA) by using forward and reverse primers which are with the tetO2 -1. By using reverse PCR, we can insert the tetO2 -1 site into the pEGFP while the plasmid produced is still in double-stranded circular form.
   2. pEGFP-loxp-km-loxp-tetO2-1 was then transformed into DH10B to greatly amplify the product by using bacterial cells
2. tetO2 -2 (DNA biding site)
   1. Reverse PCR was used to insert tetO2 -2 into pEGFP-loxp-km-loxp
   2. 2uL of 10-fold diluted pEGFP-loxp-km-loxp- tetO2 -2 was transformed into DH10B to greatly amplify the product by using bacterial cells
3. tetO2 – 3 (DNA binding site)
   1. Reverse PCR was used to insert tetO2 – 3 into pEGFP-loxp-km-loxp
   2. 2uL of 10-fold diluted pEGFP-loxp-km-loxp- tetO2 – 3 was transformed into DH10B to greatly amplify the product by using bacterial cells

1. Overlap PCR (involving 3 steps) was carried out to produce the fusion protein [tetR-HNS (any length)]
   1. PCR was carried out separately for 2 genes, tetR and HNS (full length, in this case)
      1. Primers used were as below:
      2. tetR: [forward] R-H-out-F; [reverse] R-H-tetR-R
      3. HNS (FL): [forward] R-H-tetR-F-out; [reverse] R-H(FL)-2-R

         Fusion protein genes produced separately using PCR