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From 2011.igem.org

7-18

In the morning we retrieved the DNA extracted yesterday and examined it by agarose gel electrophoresis but failed because the yield was few. We prepared the gel firstly and measured the concentration at the same time. When the concentration allows for restricted-enzyme digestion, we conducted single and double digestion and agarose gel electrophoresis separately for the original plasmid in the morning and the digested plasmid in the afternoon. The morning result is better and the reason for the unsatisfactory result in the afternoon calls for examination by further experiments.

7-19

Today we finished our practice experiment. The result is generally successful. In the morning our instructor professor Guanpin Yang came to give us some instructions about the final project of supportive loop and related knowledge on inhibitive system and some notes for experiments. Most important is that we solved the big problem of Tryptophan that we can only make one of its five protein genes express. Furthermore, the inhibitive loop is on the schedule. We plan to complete the theoretical work of it while finishing the supportive experiments. In the afternoon we met up to discuss the missions in the next step and some problems that need to solve. Today we start to apply for our part and should figure out the materials needed for connection and ligation of the loop and the inhibitive system.

Besides, we prepared 200 mL LB (within agarose) medium for eight dishes. Sterilization was done in the evening and tomorrow we will pour it into dishes.

7-20

Today we conducted the agarose gel electrophoresis for the preserved bacteria, poured the plate mediums and coated them for incubation.

At 8 a.m. we heated and poured the medium and dried them upside down in the incubator for about half a day. We conducted the enzyme digestion in the rest of time. Compared with last failed enzyme digestion, we concluded mainly three reasons: quantity of enzyme; time for reaction and the voltage and time for electrophoresis. To find out the specific reason, we set three control groups. There is one group with double quantity of enzyme; time for water bath are 1h, 4h and 6h; the voltages were set at 100V 30min. Other conditions are the same as before.

In the afternoon we arranged the following missions to every member to find out the connection and ligation of our loop. At 4:40 p.m. the latest reaction was done and we then ran the electrophoresis. Still the result is not satisfactory. Reasons are analyzed. In addition, we lined the bacteria on six plate mediums and incubated them in 37℃. From now on we start focus on the center loop. Fight!

7-21

Today we make enough LB medium for transformation of 10 parts. And we do the transformation using the preserved strains we did last time and cultivate them in the LB medium.

After the solid medium dried, we transform three parts that will be used in the our official experiment. The experiment today is inefficient and it takes a lot of time because we haven’t been divided into groups, and the old parts(for rehearsal) and the new parts(for official experiment) are mixed which is always confusing. So the organization of labor is very important.

Today we also make a lot of mistakes: 1.Forget to take the gel out of the observing box and turn off the UV after using UV to take photos of the gel. 2. Add ten times antibiotics to the LB because of wrong calculation. 3. Misoperation. It is necessary to keep order and discipline during the experiment.

7-22

We are divided into two groups: group 1 should do the transformation of the rest 7 parts of circuit 1, and inoculate the strains that we have done yesterday from solid medium to liquid medium. Group 2 is to isolate the plasmid of preserved strains that we have done yesterday, and run a gel after that.

In the evening, we try to make a big gel under the direction of a senior fellow which is used to recover the cut gel. We estimate the length of plasmid at the right location after running a gel check, so tomorrow we will run a big gel and recover the DNA fragment after that.

7-23

During the day, group 1 inoculate the 7 kinds of strains from solid medium where they have grown well to liquid medium, and isolate plasmid of 3 kinds of strains growing in the LB liquid medium. Group 2 run a gel and recover the DNA fragment, do the ligation of two fragments and the vector before running a gel to check out the recombinant plasmid. It is very confusing that there is no available band in the gel after exposure under UV.

At night, we preserve ten strains with 10 kinds of parts in it and isolate plasmid of them.

7-24

We test the concentration of the 10 kinds of plasmids that we have isolated yesterday. The concentration is extremely low. But we can see clear and bright bands at right location when we turn to the result of running gel. By asking the teacher for help, we know that we fall to remove all the ethanol during the DNA isolation which cause a big effect on the concentration but have no effect in running the gel.

PS: Take your time during running the gel in case of DNA running out of the gel!