Team:WHU-China/Notebook/Daytoday

From 2011.igem.org

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4/14~15: preparing materials: such as DH5α, pDsRed -Express Vector, amp, kana, TB, 0.85%NaCl, 0.1MCaCl2, 0.1MMg2+</br>
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4/14~15, preparing materials: such as DH5α, pDsRed -Express Vector, amp, kana, TB, 0.85%NaCl, 0.1MCaCl2, 0.1MMg2+</br>
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                 4/24: successfully obtained pDsRed-Express Vector plasmid</br>
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                 4/24, successfully obtained pDsRed-Express Vector plasmid</br>
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                 4/26~29: transform pDsRed-Express Vector plasmid into DH5αcompetent cell in order to test our experiment skills</br>
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                 4/26~29, transform pDsRed-Express Vector plasmid into DH5αcompetent cell in order to test our experiment skills</br>
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                 5/1~10: vocation
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                 5/1~10, vocation
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<h3><a href="#">6th Week</a></h3>
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<h3><a href="#">The third layer assembly</a></h3>
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Cras dictum. Pellentesque habitant morbi tristique senectus et netus
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7/30, ligated 8HⅡ-2MⅠ，4AⅠ-23LⅠ-2MⅠ-20KⅠ，15NⅠ-23LⅠ-6HⅢ，2KⅠ-16MⅣ，15NⅠ-23LⅠ-17PⅠ，2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ</br>
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et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in
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7/31, ligate 2MⅠ-23LⅠ- 4MⅠ，4OⅠ-23LⅠ-2IⅠ- 6IⅠ，14JⅠ-23LⅠ-2MⅠ- 6IⅠ</br>
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faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia
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8/1~2, ligate RLS5+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ) RLS1+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ)</br>
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mauris vel est.
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8/3, the ligation of 4AⅠ-23LⅠ-2MⅠ-20KⅠ,8HⅡ-2MⅠfailed，but we successfully gained the  4OⅠ-23LⅠ-2IⅠ- 6IⅠ，2MⅠ-23LⅠ- 4MⅠ，ligated the 2MⅠ-23LⅠ- 4MⅠ-12AⅠ
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<h3><a href="#">Re-ligate the failure products(1)</a></h3>
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Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus.
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8/4~5, re-ligated the 23LⅠ-6HⅢ，17PⅠ-23LⅠ，4AⅠ-23LⅠ-2MⅠ-20KⅠ，test RLS1+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ)，transformed new biobricks 19CⅣ，15OⅣ，19IⅣ，6JⅢ,6LⅢ，6NⅢ，16EⅠ，8LⅡ，12LⅠ，20ⅠⅠ,6NⅠ
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Class aptent taciti sociosqu ad litora torquent per conubia nostra, per
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8/6, re-ligated 14JⅠ-23LⅠ-2MⅠ- 6IⅠ，ligated 14HⅠ-2MⅠ，6JⅢ-23LⅠ，6LⅢ-23LⅠ，15NⅠ- RLS1+6，19CⅣ-23LⅠ，15OⅣ-23LⅠ，19IⅣ-23LⅠ，23LⅠ-14KⅠ，10EⅣ-23LⅠ，15MⅣ-23LⅠ，17AⅣ-23LⅠ
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inceptos himenaeos.
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8/7~10, ligated14JⅠ-23LⅠ-2MⅠ-6IⅠ，2MⅠ-14HⅠ-2MⅠ，6JⅢ-23LⅠ，6LⅢ-23LⅠ，15NⅠ-RLS1+6，19CⅣ-23LⅠ，15OⅣ-23LⅠ，19IⅣ-23LⅠ，15MⅣ-23LⅠ，17AⅣ-23LⅠ
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8/11~12, ligated 8HⅡ-2MⅠ-14HⅠ-2MⅠ，2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，use enzyme to verify the 6JⅢ-23LⅠ，6LⅢ-23LⅠ，17AⅣ-23LⅠ
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8/13~14, re-ligate 2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，ligate 2MⅠ-17AⅣ-23LⅠ，2MⅠ-6JⅢ-23LⅠ，2MⅠ-6LⅢ-23LⅠ
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8/15, re-ligate 8HⅡ-2MⅠ-14HⅠ-2MⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ，23LⅠ-14KⅠ，use PCR to amplify the 2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，2MⅠ-17AⅣ-23LⅠ
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<h3><a href="#">7th Week</a></h3>
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<h3><a href="#">Re-ligate the failure products(2)</a></h3>
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Cras dictum. Pellentesque habitant morbi tristique senectus et netus
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8/16, ligate 16EⅠ-2MⅠ-6LⅢ-23LⅠ，16EⅠ-15OⅣ，16EⅠ-17AⅣ，16EⅠ-19CⅣ，16EⅠ-15MⅣ</br>
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et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in
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8/17~18, ligate 4EⅠ-23LⅠ，RLS1+6-16EⅠ-15OⅣ，RLS1+6-16EⅠ-6LⅢ，RLS1+6-16EⅠ-17AⅣ，RLS1+6-16EⅠ-19CⅣ，RLS1+6-19CⅣ，16MⅣ-2KⅠ</br>
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faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia
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8/19~20, ligate 8HⅡ-2MⅠ-14HⅠ-2MⅠ-23LⅠ-14KⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ-4AⅠ-23LⅠ-2MⅠ-20KⅠ，15NⅠ-RLS，16EⅠ-2MⅠ-19CⅣ-23LⅠ- pSB1C3, 16MⅣ- RLS6</br>
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mauris vel est.
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8/21~22, re-ligate 4OⅠ-23LⅠ-2IⅠ- 6IⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ，23LⅠ-14KⅠ，8HⅡ-2MⅠ-14HⅠ-2MⅠ，16EⅠ-2MⅠ-19CⅣ-23LⅠ- pSB1C3，16MⅣ- RLS6</br>
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8/23, ligate 14JⅠ-23LⅠ-2MⅠ-6IⅠ-4AⅠ-23LⅠ-2MⅠ-20KⅠ，4OⅠ-23LⅠ-2IⅠ- 6IⅠ，8HⅡ-2MⅠ-14HⅠ-2MⅠ-23LⅠ-14KⅠ，16EⅠ-2MⅠ-19CⅣ-23LⅠ-pSB1C3，16EⅠ-15MⅣ, 16EⅠ-15OⅣ, 16EⅠ-17AⅣ, 16EⅠ-6LⅢ</br>
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8/24~25, 16EⅠ-15MⅣ-pSB1C3, 16EⅠ-15OⅣ-pSB1C3, 16EⅠ-17AⅣ-pSB1C3, 16EⅠ-6LⅢ-pSB1C3，16EⅠ-2MⅠ-19CⅣ-23LⅠ-pSB1C3, RLS-18OⅠ, RLS-18MⅠ, RLS-2GⅡ,k360123-k360126
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Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus.
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Class aptent taciti sociosqu ad litora torquent per conubia nostra, per
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inceptos himenaeos.
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Revision as of 03:21, 2 October 2011

其他

Transform pDsRed-Express Vector plasmid

4/14~15, preparing materials: such as DH5α, pDsRed -Express Vector, amp, kana, TB, 0.85%NaCl, 0.1MCaCl2, 0.1MMg2+
4/24, successfully obtained pDsRed-Express Vector plasmid
4/26~29, transform pDsRed-Express Vector plasmid into DH5αcompetent cell in order to test our experiment skills
5/1~10, vocation

Modified the procedures of competent cell production and successfully boomed the results of transformation

5/11~26, use enzyme SpelⅠto digest pDsRed-Express Vector plasmid
5/27, use XbalⅠto digest pDsRed-Express Vector plasmid
5/28, use EcoRⅠto digrst pDsRed-Express Vector plasmid
5/29~6/1, use Pst1 and EcoRⅠto digest pDsRed-Express Vector6/2~15，produce the competent cell, use T4 ligase to join DNA fragments, and conduct the transformation..During those days, we modified the procedures of competent cell production and successfully boomed the results of transformation to 10^7
6/16~7/7, too busy with the school stuff

Successfully transformed pSB1A3, pSB1C3 and biobricks 15NI, 6AI

7/8~9, pick up the procedures of transformation after the break. At the meantime, use PstI and EcoRI to digest the pDsRed-Express Vector, conduct the gel extraction and join the DNA fragments overnight
7/10, transform the products of ligation
7/11, tranform the plasmid of pSB1A3, pSB1C3 and biobrick 15NI, 6AI
7/12, re-transform pSB1C3, biobricks 15NI, 6AI
7/13, extract the plasmid of 15NI, 6AI, use EcoRI and SpeI to digest the 15NI plasmid, use EcoRI and XbalI to digest the plasmid of 6AI
7/14, re-transform the pSB1A3 and the biobricks of 15NI 6AI due to the mistakenly omitting steps of culturing E. Coli with these plasmids

Transformed Single biobricks

7/15~17, transform the plasmids of 23LⅠ,6HⅢ，17PⅠ,2KⅠ,16MⅣ，2MⅠ, 13JⅡ，20FⅠ,8HⅡ，14HⅠ,14KⅠ,20KⅠ,4AⅠ,6IⅠ,14JⅠ,12AⅠ,4MⅠ,2IⅠ,4OⅠ At the meantime, digest the 4AⅠ, 4OⅠ, 4MⅠ with EcoRI and SpelⅠ, 23LⅠ with EcoRI and XbalⅠ, then conducting ligation digest the 6HⅢ and 17PⅠwith EcoRⅠ and SpelⅠ, with an unexpected 3-band result, owing to the higher than normal plasmid concentration.
7/18, transform the biobricks of 8HⅡ,14KⅠ,20 KⅠ, 6IⅠ, 12AⅠ, gaining the ligation products of 23LⅠ-4AⅠ，23LⅠ- 4OⅠ，23LⅠ- 4MⅠ23LⅠ-6HⅢ, re-digest 6HⅢ with EcoRⅠ and SpelⅠ. 7/19, transform the biobricks of 16MⅣ，20FⅠ，14HⅠ，14JⅠ，13JⅡ, use gal extract to gain the product. 7/20, use the EcoRⅠand SpelⅠto digest 4AⅠ, 4OⅠ, 4MⅠ,use EcoRⅠand XbalⅠto digest 23L，in the meantime we use enzyme digest to test our product 4AⅠ, 4OⅠ, 4MⅠ，20 KⅠ, 6IⅠ, 12AⅠ，gain our ligation product 23LⅠ- 4MⅠ，23LⅠ-6HⅢ

The second layer assembly

7/21, complete the ligation of 23LⅠ-4AⅠ，23LⅠ- 4OⅠ，23LⅠ- 4MⅠ，23LⅠ- 14KⅠ
7/22, transform the biobricks of 6A and 15N，complete the ligation of 20 FⅠ-2MⅠ，13JⅡ-2M.
7/23~28, Focused on the ligation of biobricks . Till 7/28 we have successfully ligate the 23LⅠ- 4MⅠ，23LⅠ- 4OⅠ，23LⅠ-14KⅠ，20FⅠ-2MⅠ，13JⅡ-2MⅠ，23LⅠ-14JⅠ，23LⅠ-6HⅢ，4AⅠ-23LⅠ-2MⅠ，20 FⅠ-2MⅠ-23LⅠ，and 17PⅠ-23LⅠ
7/29, ligated the 14JⅠ-23LⅠ-2MⅠ，4OⅠ-23LⅠ-2IⅠ，14HⅠ-2MⅠ

The third layer assembly

7/30, ligated 8HⅡ-2MⅠ，4AⅠ-23LⅠ-2MⅠ-20KⅠ，15NⅠ-23LⅠ-6HⅢ，2KⅠ-16MⅣ，15NⅠ-23LⅠ-17PⅠ，2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ
7/31, ligate 2MⅠ-23LⅠ- 4MⅠ，4OⅠ-23LⅠ-2IⅠ- 6IⅠ，14JⅠ-23LⅠ-2MⅠ- 6IⅠ
8/1~2, ligate RLS5+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ) RLS1+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ)
8/3, the ligation of 4AⅠ-23LⅠ-2MⅠ-20KⅠ,8HⅡ-2MⅠfailed，but we successfully gained the 4OⅠ-23LⅠ-2IⅠ- 6IⅠ，2MⅠ-23LⅠ- 4MⅠ，ligated the 2MⅠ-23LⅠ- 4MⅠ-12AⅠ

Re-ligate the failure products(1)

8/4~5, re-ligated the 23LⅠ-6HⅢ，17PⅠ-23LⅠ，4AⅠ-23LⅠ-2MⅠ-20KⅠ，test RLS1+6(2KⅠ-16MⅣ-2MⅠ-13JⅡ-2MⅠ-20 FⅠ-23LⅠ)，transformed new biobricks 19CⅣ，15OⅣ，19IⅣ，6JⅢ,6LⅢ，6NⅢ，16EⅠ，8LⅡ，12LⅠ，20ⅠⅠ,6NⅠ 8/6, re-ligated 14JⅠ-23LⅠ-2MⅠ- 6IⅠ，ligated 14HⅠ-2MⅠ，6JⅢ-23LⅠ，6LⅢ-23LⅠ，15NⅠ- RLS1+6，19CⅣ-23LⅠ，15OⅣ-23LⅠ，19IⅣ-23LⅠ，23LⅠ-14KⅠ，10EⅣ-23LⅠ，15MⅣ-23LⅠ，17AⅣ-23LⅠ 8/7~10, ligated14JⅠ-23LⅠ-2MⅠ-6IⅠ，2MⅠ-14HⅠ-2MⅠ，6JⅢ-23LⅠ，6LⅢ-23LⅠ，15NⅠ-RLS1+6，19CⅣ-23LⅠ，15OⅣ-23LⅠ，19IⅣ-23LⅠ，15MⅣ-23LⅠ，17AⅣ-23LⅠ 8/11~12, ligated 8HⅡ-2MⅠ-14HⅠ-2MⅠ，2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，use enzyme to verify the 6JⅢ-23LⅠ，6LⅢ-23LⅠ，17AⅣ-23LⅠ 8/13~14, re-ligate 2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，ligate 2MⅠ-17AⅣ-23LⅠ，2MⅠ-6JⅢ-23LⅠ，2MⅠ-6LⅢ-23LⅠ 8/15, re-ligate 8HⅡ-2MⅠ-14HⅠ-2MⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ，23LⅠ-14KⅠ，use PCR to amplify the 2MⅠ-19CⅣ-23LⅠ，2MⅠ-15MⅣ-23LⅠ，2MⅠ-15OⅣ-23LⅠ，2MⅠ-17AⅣ-23LⅠ

Re-ligate the failure products(2)

8/16, ligate 16EⅠ-2MⅠ-6LⅢ-23LⅠ，16EⅠ-15OⅣ，16EⅠ-17AⅣ，16EⅠ-19CⅣ，16EⅠ-15MⅣ
8/17~18, ligate 4EⅠ-23LⅠ，RLS1+6-16EⅠ-15OⅣ，RLS1+6-16EⅠ-6LⅢ，RLS1+6-16EⅠ-17AⅣ，RLS1+6-16EⅠ-19CⅣ，RLS1+6-19CⅣ，16MⅣ-2KⅠ
8/19~20, ligate 8HⅡ-2MⅠ-14HⅠ-2MⅠ-23LⅠ-14KⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ-4AⅠ-23LⅠ-2MⅠ-20KⅠ，15NⅠ-RLS，16EⅠ-2MⅠ-19CⅣ-23LⅠ- pSB1C3, 16MⅣ- RLS6
8/21~22, re-ligate 4OⅠ-23LⅠ-2IⅠ- 6IⅠ，14JⅠ-23LⅠ-2MⅠ-6IⅠ，23LⅠ-14KⅠ，8HⅡ-2MⅠ-14HⅠ-2MⅠ，16EⅠ-2MⅠ-19CⅣ-23LⅠ- pSB1C3，16MⅣ- RLS6
8/23, ligate 14JⅠ-23LⅠ-2MⅠ-6IⅠ-4AⅠ-23LⅠ-2MⅠ-20KⅠ，4OⅠ-23LⅠ-2IⅠ- 6IⅠ，8HⅡ-2MⅠ-14HⅠ-2MⅠ-23LⅠ-14KⅠ，16EⅠ-2MⅠ-19CⅣ-23LⅠ-pSB1C3，16EⅠ-15MⅣ, 16EⅠ-15OⅣ, 16EⅠ-17AⅣ, 16EⅠ-6LⅢ
8/24~25, 16EⅠ-15MⅣ-pSB1C3, 16EⅠ-15OⅣ-pSB1C3, 16EⅠ-17AⅣ-pSB1C3, 16EⅠ-6LⅢ-pSB1C3，16EⅠ-2MⅠ-19CⅣ-23LⅠ-pSB1C3, RLS-18OⅠ, RLS-18MⅠ, RLS-2GⅡ,k360123-k360126

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