Team:GZHS-United/Parts

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Parts

First, we transformed the vector pSB1C3 into DH5αand cultured over night for amplification. Then, we extracted pSB1C3 from DH5αand cut them using two restriction enzymes XbaI and PstI. As a results, the vector pSB1C3 would end up with two ends, XbaI and PstI. Then, electrophoresis was used, and the vector pSB1C3 was excised from the gel.

After processing the pSB1C3, we then start processing the target gene BmK IT. We did condom optimization on wild type BmK IT to remove the illegal restriction endonuclease sites and make it express better in E.coli, and synthesized it with the help of a company. The company provided us pUC57 plasmid that contains the optimized BmK IT fragment.

We used two rounds of PCR to extract the BmK IT from pUC57 and attach the required restriction endonuclease sites to its terminal. The first round of PCR is used to add two restriction enzyme cutting sites XbaI and SpeI on our gene BmK IT. The electrophoretic result showed that the BmK IT is extracted.

Then, the second round of PCR add another cutting site PstI. We use the first round PCR product as our template to amplify the BmK IT, the electrophoretic result proofed that we obtained large amount of pure BmK IT fragment.

After these two rounds of PCR, we obtained our target gene segment BmK IT with two restriction enzyme cutting sites XbaI and PstI. After that, we use the restriction enzyme XbaI and PstI to create the sticky ends in both sides. Then they were put under 80℃ for 10min to inactivate the restriction enzyme.

Then, we mixed the opened pSB1C3 and the BmK IT together and add T4 ligase. They were placed under 16℃ overnight.

On the following day, the recombinant plasmids were transformed into DH5α and cultured for 17h.

Then we used colony PCR to examine whether BmK IT were successfully linked into pSB1C3, using the BmK IT after the first round of PCR as the control group. After using electrophoresis to test the PCR products, we found that the results are correct.

We then extracted the plasmids from DH5α and resolved them in ddH2O. We added the solution on the DNA submission plate and placed it on the lab bench to air-dry to collect the plasmids powder.