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From 2011.igem.org

8-8

We came to the lab at 16:00. We arrange to transform the linked 3-18, 11-16, 14-19. At the same time, 活化4, 5 and 11, 18 and 45 and inoculated them into LB liquid media. Later we activated 4, 5, 11, 4-5 and culture them. .In the night, we coated the plate mediums.

When Team Two were doing these operations, Team One were doing something about competent cells but they all failed, the reason of which was not clear. Team Three assisted Team One to link part 4 and part 5 . But they mistakenly put the linked ones into an environment of 37℃ (It should be 42℃). After a long time, pollution was found together with the colony.

8-9

In the morning we put the plate into a 4℃ refrigerator. Only 14-19 and puc19 were successful. 3-18 and 11-16 were not. In the morning, we planned to extract plasmid from 4, 5, 18, 11, 4-5.

This time, we were going to use the 3A method from the iGEM website to process 4 and 5 making it possible that we could get the two parts and link them with the carrier. 3, 18 and 11, 16 were processes using the common method. We did enzyme digestion again.

In the afternoon, we cultured and shook 14-19. We also tried to pick a few colonies to shake and culture. 2-6, which was reserved earlier, was together shaken and cultured. We await extracting the plasmid tomorrow.

In the evening, we did Gel electrophoresis to Group 3, 4, 5, 11, 16, 18. The latter part of them did not show anything. Then 4, 5 and plasmid were linked to 3A. 3, 11 were recycled using the kit. Carelessly, we confused 3 and 11. Double enzyme digestion through the night to 16 and 18.

8-10

At about 10, we transformed the 4-5 by 3A. We also extracted plasmid shaken and cultured yesterday. Again, we did electrophoresis to the part that we had failed to verify yesterday. We made it this time, but band 18 was a little dark. We then used the same method to figure out which was 3 and 11.

8-11

In the morning, we did double enzyme digestion to the extracted plasmid 12&19 (1, 2, 3, 4, 5), 4&5, 2&6 and ran gel electrophoresis. As a result, 14&19 band overlapped because of their similar formula weight and could not decide whether it was OK. 5&4 was a failure. So did 2&6. We can only depend on 3A linkage.

Tan suggested that we conducted single enzyme digestion. So we did this in the afternoon. 14&19 (1, 2, 3, 4, 5). When we conducted the gel electrophoresis we add the 14 plasmid and digested 14 plasmid as a control group. It was successful. 14&19 and 2&6 were both successful.

In the evening, we did double enzyme digestion to 2-6&3, 18 &3, 11&16. And tomorrow we are going to do 3A linkage through the night.

By the way, in the afternoon, we put the 5&4 colony in the LB liquid media and extracted the plasmid the next day.