Difference between revisions of "Team:NWU-CHINA/Notebook"

We noticed Igem competition and get acquainted with synthesis biology.

Our team, NWU-CHINA, had been organized. Then we find our PI and confirmed our project. Our PI gave us a lot of advice about following experiment.

We accepted foundational lab training and learned necessary experiment operation.

We cleared Interlab Study requirement, RFC and 3A assembly protocol. Meanwhile, we found a lot of reference paper to find theory supporting our experiment.

We finished our First Interlab Study

Using Pak1900 to construct RFP vector and transferring constructed vector into P.a to verify expression of RFP in P.a .The detail protocol is as follows:

1. We got RFP sequence from registry and cloned RFP gene by PCR. Then we separated cloned RFP
2. gene by agarose gel electrophoresis and purified the gene by gel slices.
3. We used pAK1900 as our vector. Then we used BamH I and Sac I to digest pAK1900 and purified gene for 5 hours.
4. Then we used T4 DNA Ligase to ligated our vector and RFP gene at 4 ℃ for 24 hours
5. We transferred our vector into E.coli DH5&alpha competent cell to get more vector clones. Our transferring protocol is as follows:
1. Mix 4ul vector and 50ul competent cells in 1.5mL centrifuge tube at 0 ℃ for 30 min.
2. Hot shock for 90 seconds at 42℃ .
3. Keep competent cell at 0℃ for 4-5 min, then add 950&microL Soc media into the tube.
4. Incubate at 37 ℃ for 2 hours shaking at 220rpm
5. Centrifuge at 6000rpm for 1min. After centrifugation, 900&microL supernatant is discarded, then resuspended cells, spread the culture on plate with antibiotic 100&microg/&microL TC , incubate at 37℃ for 16 hours.
6. Select several colonies, purified them on another selective plate, incubate at 37℃ for 12 hours.
6. Incubate purified cultures with 5mL LB media and 5ul 5mg/&microL TC for 5 hours.
7. Using Plasmid minipreparation kit to extract the vector.

We had done our second Interlab Study. Meanwhile, we used PCR to clone GntR and AlkB2 gene. And ligated them on constructed RFP vector. Then we separated cloned gene by agarose gel electrophoresis and purified the gene by gel slices.

1. We used Hind I, Hind II, Sal I, Hind III and Xba I, Spe I and Sal I digest pAK1900, GntR, AlkB2 for 5 hours respectively.
2. Then we used T4 DNA Ligase to ligated them at 4℃ for 24 hours.
3. Then we cloned our vector in DH5&alpha competent cells.
4. Sequence our gene to verify our vector.

We constructed our submission parts.