Team:NCTU Formosa/notes

From 2013.igem.org

(Difference between revisions)
(→About the notes)
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<div class="week">
<div class="week">
<div class="day">
<div class="day">
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<div class="daybar"><p>11</p></div>
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<div class="daybar"><p>24</p></div>
-
<div class="dots">
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</div>
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<div class="open">
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</div>
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</div>
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<div class="day">
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<div class="daybar"><p>12</p></div>
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-
<div class="dots">
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-
</div>
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-
 
+
-
<div class="open">
+
-
</div>
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-
 
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-
</div>
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<div class="day">
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<div class="daybar"><p>13</p></div>
+
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>mini-prep of cultivated AlsS, pSB1K3, pSB1A3, and pSB1C3 E.coli  (G2)</p></li>
 
</ul>
</ul>
-
</div>
 
-
 
-
</div>
 
-
<div class="day">
 
-
<div class="daybar"><p>14</p></div>
 
-
<div class="dots">
 
-
<ul>
 
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
-
</ul>
 
-
</div>
 
-
 
-
<div class="open">
 
-
<ul>
 
-
<li class="green e2"><p>digestion : [Zif268] ES &amp; [AlsS] XP &amp; [pSB1C3] EP  (G2)</p></li>
 
-
<li class="green e2"><p>ligation : insert[Zif268] ES &amp; [AlsS] XP/Vector[pSB1C3] EP  (G2)</p></li>
 
-
</ul>
 
-
</div>
 
-
 
-
</div>
 
-
<div class="day">
 
-
<div class="daybar"><p>15</p></div>
 
-
<div class="dots">
 
-
<ul>
 
-
</ul>
 
-
</div>
 
-
 
-
<div class="open">
 
-
<ul>
 
-
</ul>
 
-
</div>
 
-
 
-
</div>
 
-
<div class="day">
 
-
<div class="daybar"><p>16</p></div>
 
-
<div class="dots">
 
-
<ul>
 
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
-
</ul>
 
-
</div>
 
-
 
-
<div class="open">
 
-
<ul>
 
-
<li class="green e2"><p>Transformation of B0034</p></li>
 
-
<li class="green e2"><p>Cultivation of transformed E.coli on LBA plate</p></li>
 
-
</ul>
 
-
</div>
 
-
 
-
</div>
 
-
<div class="day brn">
 
-
<div class="daybar"><p>17</p></div>
 
-
<div class="dots">
 
-
</div>
 
-
 
-
<div class="open">
 
-
</div>
 
-
 
-
</div>
 
-
</div>
 
-
<!---------------------------------------- week start ---------------------------------------->
 
-
<div class="week">
 
-
<div class="day">
 
-
<div class="daybar"><p>18</p></div>
 
-
<div class="dots">
 
-
</div>
 
-
 
-
<div class="open">
 
</div>
</div>
</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>19</p></div>
+
<div class="daybar"><p>25</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Transformation of Zif268+AlsS+pSB1C3 in DH5-alpha  (G2)</p></li>
 
-
<li class="green e2"><p>Point mutation HivC (PCR, DPN1 digest, TA cloning, transform) (G2)</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>20</p></div>
+
<div class="daybar"><p>26</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>PCR of point mutation HivC  (G2)</p></li>
 
-
<li class="green e2"><p>PCR of insert fragment [Zif268+AlsS]  (G2)</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>21</p></div>
+
<div class="daybar"><p>27</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>electrophoresis of insert fragment [Zif268+AlsS]----NOT OK  (G2)</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>22</p></div>
+
<div class="daybar"><p>28</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Single colony isolation from pLac LBA plate, and cultivation of pLac E.coli in liquid LBA</p></li>
 
-
<li class="green e2"><p>Single colony isolation from pSB1K3 LBK plate, and cultivation of pSB1K3 E.coli in liquid LBK</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>23</p></div>
+
<div class="daybar"><p>29</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>mini-prep of cultivated pLac &amp; pSB1K3 E.coli</p></li>
+
<li class="green e2"><p>Transformation of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) and cultivation  on three LB plates.</p></li>
 +
<li class="green e2"><p>transformation of PompC and
 +
cultivation  on LB-A plate</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day brn">
<div class="day brn">
-
<div class="daybar"><p>24</p></div>
+
<div class="daybar"><p>30</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
<li class="caldot green"></li>
<li class="caldot green"></li>
</ul>
</ul>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green"><p>digestion : [pSB1K3] EP</p></li>
+
<li class="green e3"><p>Single colony isolation from three plates and cultivation them in liquid LB</p></li>
 +
<li class="green e3"><p>Single colony isolation from PompC LB-C plate and cultivation in liquid LB-A</p></li>
 +
<li class="green e3"><p>mini-prep of cultivated PompC E.coli</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
</div>
</div>
 +
<!---------------------------------------- week end ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
<!---------------------------------------- week start ---------------------------------------->
<div class="week">
<div class="week">
<div class="day">
<div class="day">
-
<div class="daybar"><p>25</p></div>
+
<div class="daybar"><p>31</p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
 +
<li class="caldot green"></li>
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
 +
<li class="green e4"><p>Transformation of RBS  B0030+PSB1A3&B0034+PSB1A3 and cultivation on LB-A plate.</p></li>
 +
<li class="green e4"><p>Mini-prep for cultivated  ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) PCR of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar).</p></li>
 +
<li class="green e4"><p>Electrophoresis of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) ──NOT OK.</p></li>
 +
<li class="green e4"><p>PCR  of insert fragment
 +
[PompC+psB1A2]</p></li>
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>26</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>27</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>28</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>29</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>Single colony isolation from Zif268 LBA plate, and cultivation of Zif268 E.coli in liquid LBA</p></li>
 
-
<li class="green e2"><p>Single colony isolation from AlsS LBA plate, and cultivation of AlsS E.coli in liquid LBA</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day">
<div class="day">
-
<div class="daybar"><p>30</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
-
<li class="caldot green"></li>
 
-
<li class="caldot green"></li>
 
</ul>
</ul>
</div>
</div>
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<div class="open">
<div class="open">
<ul>
<ul>
-
<li class="green e2"><p>mini-prep of cultivated Zif268 Ecoli &amp; AlsS Ecoli</p></li>
 
-
<li class="green e2"><p>digestion :[Zif268] ES/[AlsS] XP</p></li>
 
</ul>
</ul>
</div>
</div>
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</div>
</div>
<div class="day brn">
<div class="day brn">
-
<div class="daybar"><p>31</p></div>
+
<div class="daybar"><p></p></div>
<div class="dots">
<div class="dots">
<ul>
<ul>
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</div>
</div>
<!---------------------------------------- week end ---------------------------------------->
<!---------------------------------------- week end ---------------------------------------->
-
</div>
 
</div>
</div>
</div>
</div>
 +
</div>
<!---------------------------------------- cal ends ---------------------------------------->
<!---------------------------------------- cal ends ---------------------------------------->
</div>
</div>

Revision as of 17:26, 20 September 2013

Notes

The experimental log that records down the purpose, method, result and date of each experiment that we have conducted.

About the notes

Our notes are the abbreviations of the experimental process, including titles, genes we transformed, plasmids we used, and resistances on the plasmids. We categorized these notes by date. Moreover, team members followed the iGEM protocol in each experimental step in order to get better experimental efficiency and the results we had expected.

March 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

24

25

26

27

28

29

  • Transformation of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) and cultivation on three LB plates.

  • transformation of PompC and cultivation on LB-A plate

30

  • Single colony isolation from three plates and cultivation them in liquid LB

  • Single colony isolation from PompC LB-C plate and cultivation in liquid LB-A

  • mini-prep of cultivated PompC E.coli

31

  • Transformation of RBS B0030+PSB1A3&B0034+PSB1A3 and cultivation on LB-A plate.

  • Mini-prep for cultivated ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) PCR of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar).

  • Electrophoresis of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) ──NOT OK.

  • PCR of insert fragment [PompC+psB1A2]

April 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]

2

  • transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate

3

4

  • digestion : [pSB1K3] EP

  • digestion : [pSB1K3] EP

5

6

7

  • PCR of insert fragment [Zif268+AlsS] OK

  • DNA Sequencing OK

  • ligation : point mutation HivC

  • TA cloning : point mutation HivC

8

9

  • Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K

10

  • mini-prep of cultivated Zif268+ AlsS E. coli

  • transformation of point mutation HivC and cultivation on LB-A plate

  • transformation of B0034 and cultivation on LB-A plate

11

12

  • Single colony isolation from HivC LB-A plate, and in liquid LB-A

  • Single colony isolation from B0034 LB-A plate, and in liquid LB-A

13

  • mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli

14

  • digestion : Zif268+AlsS [XP]

15

16

17

  • Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A

18

  • mini-prep of cultivated ilvD E. coli

  • digestion : ilvD [EP] & pSB1K3 [EP]

  • transformation of 37℃ RBS and cultivation on LB-C plate

19

  • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

20

  • digestion : B0034 [SP]

  • gel extraction

  • ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]

  • mini-prep of cultivated Hivc E. coli

21

  • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

  • transformation of HivC and cultivation on LB-A plate

22

  • PCR of insert fragment [B0034+Zif268+AlsS] OK

  • DNA Sequencing NOT OK

  • digestion : B0034 [SP] & Zif268+AlsS [XP]

  • gel extraction

  • ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]

  • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

  • Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

  • SCI from ilvD LB-K plate, and cultivation in liquid LB-K

23

  • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

  • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

  • mini-prep of cultivated B0034 and ilvD E. coli

  • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

24

  • mini-prep of cultivated HivC E. coli

  • digestion : B0034 [SP] & ilvD [XP]

25

  • digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]

  • ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]

  • ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]

26

  • transformation of HivC+ilvD and cultivation on LB-A & LB-C plate

27

  • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

28

  • transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate

  • PCR of insert fragment [HivC+ilvD] OK

  • DNA sequencing NOT OK

29

  • Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C

30

  • mini-prep of cultivated & pSB1C3 E. coli

May 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • digestion : pLac [EP] & pSB1K3 [EP]

  • ligation :insert pLac [EP]/Vector pSB1K3 [EP]

  • transformation of pLac+pSB1K3 and cultivation on LB-K plate

2

  • PCR of insert fragment [pLac+pSB1K3] OK

  • DNA Sequencing OK

  • transformation of B0034_new and cultivation on LB-A plate

  • Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

3

  • mini-prep of cultivated B0034 E. coli

  • digestion : B0034 [SP]

  • ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]

  • transformation of B0034+Zif268+AlsS

  • cultivation on LB-A plate

4

  • PCR of insert fragment [B0034+Zif268+AlsS]-----self ligation

  • transformation of 37℃ RBS and cultivation on LB-C plate

5

6

7

8

  • transformation of 37℃ RBS and cultivation on LB-Kplate

9

  • Single colony isolation from 37℃ RBS LB-K plate, and cultivation in liquid LB-K

10

  • digestion : B0034 [ES] & pSB1C3 [EP]

  • mini-prep of cultivated 37℃ RBS E. coli

11

  • ligation :insert B0034 [ES] & pSB1C3 [EP]/Vector pSB1C3 [EP]

  • transformation of B0034+Zif268+AlsS and cultivation on LB-C plate

12

  • digestion : pLac [ES] & pLac [SP]

  • digestion : B0034 [SP] Kr (gel extraction) , B0034 [SP] Ar (gel extraction) , 37℃ RBS [XP] & HivC [XP]

  • ligation : (1. 2 parts) insert HivC [XP], Vector B0034+pSB1K3 [SP]

  • ligation : (2. 3 parts) insert HivC [XP], Vector B0034+pSB1A3 [SP]

13

14

15

  • PCR of insert fragment [B0034+Zif268+AlsS] OK

  • DNA Sequencing OK

  • transformation of B0034+ HivC and cultivation on LB-A & LB-K plate

16

17

18

  • Single colony isolation from pSB1A3 LB-A plate, and cultivation in liquid LB-A

  • Single colony isolation from B0034+Zif268+AlsS LB-C plate, and cultivation in liquid LB-C

  • Single colony isolation from B0034+HivC LB-A & K plate, and cultivation in liquid LB-A & K

19

  • mini-prep of cultivated pSB1A3 E. coli & B0034+Zif268+AlsS

  • digestion: pSB1A3 [EP] & B0034+Zif268+AlsS [XP]

  • ligation: (1.3 parts)insert pLac [ES] & B0034+Zif268+AlsS [XP], Vector pSB1A3 [EP]

  • ligation: (2.2 parts)insert B0034+Zif268+AlsS [XP], Vector pLac+pSB1K3 [SP]

  • transformation of pLac+B0034+Zif268+AlsS and cultivation on LB-A plate & LB-K plate

  • mini-prep of cultivated B0034+HivC (Ar & Kr)

20

  • PCR of insert fragment [pLac+B0034+Zif268+AlsS] OK

  • DNA Sequencing 3 parts OK

21

  • digestion : 37℃ RBS [XP] , ilvD [ES] & pSB1C3 [EP]

22

23

  • digestion : HivC+ilvD-3,18 [EP]

  • ligation :insert ilvD[ES] & 37℃ RBS [XP], Vector pSB1C3 [EP]

24

  • Single colony isolation from pLac+B0034+Zif268+AlsS LB-A plate, and cultivation in liquid LB-A

25

  • mini-prep of cultivated pLac+B0034+Zif268+AlsS E. coli

  • transformation of ilvD+37℃ RBS and cultivation on LB-C plate

26

  • PCR of insert fragment [ilvD+37℃]-----OK

  • DNA Sequencing OK

27

28

29

30

  • Single colony isolation from PBSII+ilvC LB-K plate, and cultivation in liquid LB-K

  • ligation : insert HivC [XP], vector B0034+pSB1A3 [SP]

31

  • mini-prep of cultivated PBSII+ilvC E. coli

  • digestion : pSB1C3 [EP] & PBSII+ilvC [XP]

  • ligation : insert B0034 [ES] & PBSII+ilvC [XP], vector pSB1C3 [EP]

  • transformation of B0034+PBSII+ilvC and cultivation on LB-C plate

  • transformation of B0034+HivC and cultivation on LB-A plate

June 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • PCR of insert fragment [B0034+PBSII+ilvC] OK!

  • DNA Sequencing OK!

2

3

  • mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli

  • mini-prep of cultivated HivC+ilvD-12,20 E. coli

4

  • digestion : HivC+ilvD-12,20 [XP]

5

  • Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)

6

  • digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]

  • ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]

  • ligation : insert HivC [EP]/vector pSB1C3 [EP]

  • transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate

7

  • PCR of insert fragment [B0034+ HivC] & [HivC] OK

8

  • Design the primer for B0034+PBSII+ilvC point mutation (pm)

  • Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C

9

  • mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli

  • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR

  • ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP]

10

  • transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate

  • Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed

11

  • DNA Sequencing B0034+HivC & HivC OK

  • PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK

12

  • Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A

  • Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A

13

  • mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli

  • Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better

14

  • PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK

15

  • DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK

16

17

18

19

  • PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK

  • digestion: pLac+B0034+Zif268+AlsS [DPn1]

20

  • transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate

21

  • Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A

22

  • mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli

  • digestion : pLac+B0034+Zif268+AlsS (pm) [EP]

23

  • DNA sequencing OK

24

25

  • PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

  • digestion : B0034+ PBSII+ilvC [DPn1]

26

  • transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail

27

  • PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

28

  • digestion : B0034+ PBSII+ilvC [DPn1]

29

  • transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate

30

  • PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK

  • Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C

July 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli

  • digestion : B0034+ PBSII+ilvC (pm) [EP]

2

  • DNA sequencing OK

3

4

5

6

7

  • digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]

8

9

  • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR

  • digestion : pSB1K3 [EP]

  • ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]

  • transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate

10

  • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR

  • Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1]

  • PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK

  • SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K

11

  • mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli

  • DNA sequencing OK

  • culture condition test: activation DH5αovernight

12

  • transfer to new medium(1/100), OD0.2 start counting culture time

  • transfer to 30゜C and 27゜C

13

  • transfer to 30゜C and 27゜C

14

15

  • transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate

16

  • PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK

17

  • Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]

  • Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1]

18

19

  • transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)

20

  • PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK

  • Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A

21

  • mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli

  • Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP]

  • ligation : insert G1[ES] & G2[XP] vector pSB1C3[EP]

22

  • transformation of G1+G2,and cultivation on LB- C plate failed

23

  • ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]

24

  • transformation of G1+G2,and cultivation on LB- C plate

  • Digestion: G2’(B0034+HIVC+ilvD) [XP]

  • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

25

  • PCR of insert fragment [kivD+B0015]

  • transformation of G1+G2’, and cultivation on LB- C plate failed

26

  • Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C

  • sample and do GC

27

  • mini-prep of cultivated kivD+B0015 E. coli

28

29

  • Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]

  • Digestion: G2’ [DPn1]

30

  • transformation of G2’, and cultivation on LB- A plate

31

  • Digestion: kivD+B0015 [EP] (checking bp----failed)

  • Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A

August 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • mini-prep of cultivated G2’ E. coli

2

3

4

5

6

7

8

9

  • Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]

  • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

10

  • strain test: activation of different strains overnight

11

  • transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C

12

  • Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C

  • transfer to 27゜C

13

  • mini-prep of cultivated G1+G2’ E. coli

14

15

  • Digestion: Ptet+B0032[ES] & GliI+KivD[XP]

  • sample and do GC

16

  • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

  • Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C

17

  • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

  • mini-prep of cultivated G1+G2 E. coli

18

  • PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK

  • DNA sequencing------NOT OK

19

20

21

22

23

24

  • carbon source test: activation DH5α overnight

25

  • transfer to new medium(1/100), OD0.2 start counting culture time

26

  • culturing for 72hours, inject feeding solution per 24hours

27

  • culturing for 72hours, inject feeding solution per 24hours

28

  • transformation of DNA program, and cultivation on LB- A plate

  • culturing for 72hours, inject feeding solution per 24hours

29

  • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

  • Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C

  • sample & do GC

30

  • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

  • mini-prep of cultivated DNA program E. coli

  • Do GC

31

  • PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK

  • DNA sequencing NOT OK

Retrieved from "http://2013.igem.org/Team:NCTU_Formosa/notes"