Scheme

Procedures

1. Amplification of Biobricks
   
2. Add RBS
   
3. Add promoter
   
4. Add terminator
   

Plasmid Construction

9.29 After RBS added, all seven plasmid were cut and ligated with two promoter, J23101 and J23106 respectively.

Enzyme digestion

Ligation

To complete construction quickly, we use 3A assembly to achieve plasmid with resistant to chloramphenicol (A) and standard assembly with resistant to Ampicillin (B).

Ligation: In PCR system, 16 to ligate, 65℃ to inactive, and store at 4℃.

Transformation

1. Place 7 EP tubes of 100μL DH5α competent cells on ice from -80℃ to melt.
   
2. Transfer 50μL competent cells to 7 new sterilized EP tubes from each tubes in 1.
   
3. Add 10μL of DNA to one EP tube with competent cells respectively.
   
4. Put all EP tubes on ice for 30mins.
   
5. Incubate in water at 42℃ for 90 seconds, then immediately on ice for 2 minutes.
   
6. Add 200μL SOC broth, then put in a shaking incubator for 40 minutes at 37℃ , 220rpm.
   
7. Centrifuge at 4500rpm for 2minutes, dispose 200μL supernatant.
   
8. Resuspend competent cells and spread plates.
   

Incubate at 37

Characterization

References

1. [http://www.tiangen.com/en/?productShow/t1/4/id/32.html |TIANprep Mini Plasmid Kit]
2. [http://www.tiangen.com/en/?productShow/t1/4/id/41.html |TIANprep Midi Purification Kit]
3. |NEB Biobricks® Assembly Kit