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From 2011.igem.org

7-11

Today, three of us have moved to Lao Shan campus from Yu Shan. At the same time, we toke the plasmid and the 2011 kit to Qingdao Institute of Bioenergy and Bioprocess Technology, and stored them in 4 ℃ environment. In addition, we cleared up our bags and made some preparation for our item.

7-12

Today we went to the lab to arrange the experiment desk and browsed the equipment by the guide of researchers in the institute. Besides, we specified the procedure of our experiment to prepare for tomorrow’s work.

7-13

Today we started to conduct experiments with some part that not related to our design for practice. In the morning we made LB medium (liquid and solid), SOC liquid medium to which we did not add glucose before sterilization. In the afternoon we sterilized cultures and some other materials (three boxes with pipette tips in large, middle and small size; one bag of 1.5mL EP tubes and two 1、5ml EP tubes). In the evening, we applied 2 mL glucose solution into SOC culture through 0.22μm-filtration membrane. All the mediums are preserved in 4℃ as preparation for tomorrow’s transduction experiment.

7-14

In the morning we prepared 5 mL Kanamycin solution in an EP tube and divided it into 5 EP tubes, which were marked numbers and reserved in -20℃. As we forgot to prepare plate mediums in advance, the transduction was delayed till tomorrow. In the afternoon we heated the LB medium (within agar) to liquid state with microwave and poured it into four dishes after cooling for several seconds. We cooled three of them to solid state and placed them upside down in 32℃ incubator. Tomorrow we are going to coated the plate mediums.

7-15

The transduction experiment started in 5 p.m. We thawed a tube of competent cells on ice. After transduction, we added separately 2, 20, 200 μL of mixed solution on three plate mediums and coated them evenly. Then we placed them upside down (highlighted!) in 35℃ incubator (it should be 37℃ though, but there were no that condition at that time). After 12 to 14 hours, we will see the result at about 10 a.m. tomorrow.

7-16

Since the density of clones was not satisfactory, we started transfer experiment in the afternoon to wait for the colonies to grow. There is one point we forgot that according to the design there should be a blank control group, which should be remembered in formal experiments. In the afternoon we picked some single colonies and transferred them into LB liquid medium.

7-17

Today two team members come here from Yushan campus and one of them will stay for experiment. We preserved the transgenic cells with glycerin, which will be taken back to Yushan by our team member for synchronous experiment. Besides, we extracted plasmid DNA with standardized kit. The procedure must strictly obey the protocol. Mention the order of different reagents and control of time and quantity. Furthermore, do tenderly when mixing the solution upside down.

The DNA solution is preserved in -20℃.