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Team:KAIST-Korea/Notebook/Week3
From 2011.igem.org
Week 3
6/19 Sunday
All dry lab members
@ KIB
We developed the primerKAIST to help us run PCR. Given the target DNA and the template DNA, the program, unlike other commercial programs available to this day, considers twelve factors all known to be important in designing a primer and returns the optimum sequence of primer that can be used to run PCR. We implemented a simple graphical user interface and packed it into an executable file. We will include a link so that other teams can use it as well. The program provides primers for running regular PCR and PCR for the Gibson Assembly.
Seoneung, Sohyun, Youngjun
@ KIB
Today, we’ve done two things.
First, we continued performing the experiments to get proper mini-prep samples of the three biobricks – E0420, J37033, Q04121. For the E0420, we failed in transforming it again thereby, trying to transform it again as well as preparing another sample for the electrophoresis by treating the previous mini-prep sample of the E0420. For the J37033, the gel electrophoresis that was done in the morning didn’t show the proper bands of the J37033 and we decided to perform the electrophoresis after making three samples of J37033. For the Q04121, due to the failure of re-transformation, we again transformed it. We’re going to perform the electrophoresis for J37033 and E0420 and amplify the E0420- and Q04121-containing transformed cells.
Second, we checked if the transformation of the three construct biobricks had been achieved well. The gel electrophoresis of the six samples, two for each construct plasmid, showed perfect results, meaning that we succeeded in getting proper mini-prep samples of the three construct plasmids. We’re going to use them in 3A assembly that will be started sooner or later.
In conclusion, we continued trying to guarantee the mini-prep samples of the three problematic biobricks and have prepared the proper mini-prep samples of the three construct plasmids.
6/20 Monday
All wet lab members except Jeonghyun
@ KIB
Today, we’ve done two things. First, we replaced the three problematic biobricks, J37033, Q04121, and E0420 with other new biobricks – C0062, C0012, E0020, and R0011 (we transformed the four biobricks into the competent cells). Second, we started the experiment to investigate if 1-hour or 2-hour restriction enzyme treatment is enough in order to improve the efficiency of the experiment (the current protocol is of 10-hour).
6/21 Tuesday
All wet lab members except Jeonghyun
@ KIB
Today, we’ve done three things. First, we amplified the competent cells containing four new biobricks, C0062, C0012, E0020, and R0011, which were transformed yesterday. Second, we continued the experiment to check if 1-hour or 2-hour restriction enzyme treatment is enough and checked 2 hours is enough to cut the plasmids properly before the gel electrophoresis for check.
6/22 Wednesday
Jijung, Jooyoung, Joonhyuk, Youngjun
@ KIB
Today, we’ve done five things. First, we extracted the plasmids of the four new biobricks, C0062, C0012, E0020, and R0011. Second, we started assembling the biobricks. We began with five pairs of the biobricks, B0034+C0261, B0034+E0040, B0034+C0070, B0034+C0077, and B0034+C0079, which are corresponding to RBS+luxI, +GFP, +rhlI, +cinR, and lasR, respectively, by treating them with the restriction enzymes and the ligase (for more than 10 hours).
6/23 Thursday
Jijung, Jooyoung, Joonhyuk, Youngjun
@ KIB
Today, we’ve done three things. First, we tried comparing the results of 1-hour ligation at 20-25℃ and of 10-hour ligation at 14℃. Both experiments showed the good results although there appeared several improper bands. Second, we transformed the five assembled biobricks, B0034+C0261, B0034+E0040, B0034+C0070, B0034+C0077, and B0034+C0079, into the competent cells in order to verify the proper ligation.
6/24 Friday
Jooyoung, Joonhyuk, Youngjun
@ KIB
Today, we’ve done two things.
First, we continued working on the five pairs of the biobricks, B0034+C0261, B0034+E0040, B0034+C0070, B0034+C0077, and B0034+C0079. We all of the plates except for B0034+C0079 contained colony of the transformed competent cells. However, all of the colonies were red, which meant either the self-ligation or no cut of the construction plasmids. Thus, we again treated the five pairs with the restriction enzymes and the ligases and tried checking the bands through the gel electrophoresis. But still, it didn’t show the proper bands although the absence of good experiment skill seems to be the reason this time.
Second, we started working on another four pairs of the biobricks, B0034+E0020, B0034+C0062, B0034+E1010, and B0034+E0430. We tried checking the bands after treating them with the restriction enzymes and the ligases. But we couldn’t get good results.
In summary, we continued assembling the five pairs of the biobricks, started assembling another four biobricks and failed to get good results on the gel electrophoresis check.
