Team:NTU-Taida/Notebook/Journal/September

September Journal
=2013.9.2-9.7=
==2013.09.03==
<html>
<ol>
<li> Sequencing: send 18 samples includes:</li>
ORhlB5 40  F, ORhlB5 40  R, OLasCb 32  F, OLasCb 32  R, CEA 2  F, CEA 2  R, CEA 2  GFP, RBS-pqsR-tt 7  F, BCinCg 6  F, BCinCg 6  R, BCinB5 12  F, BCinB5 12  R, C1 BCI 29  F, C1 BCI 29  R, ABC Rhl b 6  F, ABC Rhl b 6  R, ABC Rhl l 1  F, ABC Rhl l 1  F.
</ol>
<li>Ligation</li>
<ol>
<li>BLas-Cg (=BLas-E1)</li>
<li>BLas-Cm (=BLas-B5)</li>
<li>BLas-Cb</li>
</ol>
</ol>
</html>
==2013.09.04==
<html>
<ol>
<li>Still no competent cells.</il>
<li>Check: total 24 samples</li>
Result:
[[File:NTU-Taida-journal-September-1.jpg|700px|thumb|center]]
18A=Pc18A-RBS-CinR
VERY strange bands!!!
[[File:NTU-Taida-journal-September-2.jpg|700px|thumb|center]]
18C=Pc18C-RBS-CinR

<li>Mini</li>
<ol>
<li>Pc18C-ACin 3,4</li>
<li>Pc18A-RBS-CinR 2,3</li>
<li>BCinBCI 3,4</li>
<li>BLasBCI 2,3</li>
<li>pCIB5 3</li>
</ol>

<li>Digestion</li>
<ol>
<li>BLas(5): E/S</li>
<li>E1-PcD(4): E/X</li>
<li>E1(1): X/P</li>
<li>ApqsR(7): X/P</li>
</ol>

<li>Ligation</li>
<ol>
<li>Blas-[E1-PcD] </li>
<li>pCI-E1</li>
<li>Pc-[RBS-pqsR-tt]</li>
</ol>
</ol>

Ligation at 16 degree overnight
</html>

==2013.09.05==
One day more. Another day, another destiny. This never ending road of cloning~
<html>
<ol>
<li>Digestion </li>
<ol>
<li> pCIB5(3): X/P </li>
<li> pCIB5(3): E/S </li>
<li> C1BCI(29): S/P </li>
<li> BLasBCI(2): S/P </li>
<li> B0015 8/22: E/X </li>
<li> Pc18C ACin(4): E/S </li>
<li>Pc18A-RBS-CinR(2): E/S </li>
</ol>

Since we forgot which was B0015 when gel electrophoresis > <, we use 2 B0015 when ligation.

<li> Ligation </li>
<ol>
<li> PcAC1BCI-pCIB5 (=NRhlB5 αβγ) </li>
<li> pCI-B5-PcAC1BCI (=NRhlB5 γαβ) </li>
<li> C1BCI-pCIB5</li>
<li> BLasBCI-pCIB5</li>
<li> Pc18C ACin-B0015 8/22 (=18CACin) </li>
<li> Pc18A ACin-B0015 8/22 (=18A ACin) </li>
<li> C1-B5</li>
<li> [RBS-CinR]-B0015 8/22 (=ACin 1) </li>
<li> [RBS-CinR]-B0015</li>
</ol>
Ligation at 16 degree overnight

<li> Transformation: total 7 plates</li>
<ol>
<li>BLas-[E1-PcD]  Amp </li>
<li> pCI-E1  Amp </li>
<li> PcApqsR (=Pc-RBS-pqsr-tt)  Amp  Chl</li>
<li> BLas-Cg  Amp </li>
<li> BLas-Cm  Amp </li>
<li> BLas-Cb  Amp </li>
</ol>

<li> Sequencing: 13 samples include: </li>
OB5Rhl(1)  F, OB5Rhl(1)  R, OCbLas(5)  F, OCbLas(5)  R, E1-PcD(4)  F, E1-PcD(4)  R, pCIB5(3)  F, BCinBCI(4)  F, BCinBCI(4)  R, BLasBCI(2)  F, BLasBCI(2)  R, Pc18C ACin(4)  F, Pc18A-RBS-CinR  F

<li>NTU-Taiwan visited us~~~</li>

<li> We have new competent cells! </li>
</ol>

</html>
==2013.09.06==

I dreamed a dream when time gone by, when hope was high and things worked (seemed) smoothly
<html>
<ol>
<li> Go to Center of Genomic Medicine to have a short presentation and course.</li>

<li> Colony PCR: 30 tubes</li>
<ol>
<li> BLas-[E1-PcD] </li>
<li> pCI-E1</li>
<li> PcApqsR (=Pc-RBS-pqsr-tt) </li>
<li> BLas-Cg</li>
<li> BLas-Cm</li>
<li> BLas-Cb</li>
</ol>
Used wrong program so redid it. Run gel electrophoresis next week.

<li> Transformation: 9 plates</li>
<ol>
<li>  NRhlB5 αβγ  Amp</li>
<li>  NRhlB5 γαβ  Amp</li>
<li>  C1BCI-pCIB5  Amp</li>
<li>  BLasBCI-pCIB5  Amp</li>
<li>  Pc18C ACin  Amp</li>
<li>  Pc18A ACin  Chl</li>
<li>  C1-B5  Amp</li>
<li>  ACin 1  Amp</li>
<li>  ACin 2  Amp</li>
</ol>

Transform at room temperature over weekend.
</ol>
</html>
=2013.09.09-14=
==2013.09.09==
<html>
Crying alone is not allowed, not in my castle on the cloud.
<ol>
<li>Digestion: all use X/P to cut, in order to change backbone to pSB1C3 </li>
</ol>
<ol>
<li>OB5Rhl 1</li>
<li> OCbLas 5</li>
<li> ABC Rhl b 6</li>
<li> ABC Rhl l 1</li>
<li> OLasCb 32</li>
<li> ORhlB5 40</li>
<li> ApqsR 7</li>
<li> ABC Rhl m 1</li>
<li> ACE 2</li>
<li> Cb 3</li>
<li> Cl 1</li>
<li> AbaR 3</li>
<li> CEA 2</li>
</ol>

Cb didn’t have correct band, so didn’t use.
1, 2, 5, 6 are about 2K, alike vector length. Not very sure what we cut were pure inserts.

<li> Ligation</li>
<ol>
<li> 1~13 except Cb, total 12 samples. </li>
<li> Since we forgot to cut pSB1C3 at X/P, we were a little delay. </li>
</ol>

<li> Transformation (at CGM): 12 plates, all with Chl. Use half ligation products with 50ul competent cell. Leave half ligation O/N at 4 degree. </li>
<ol>

<li> OB5Rhl 1
<li> OCbLas 5 </li>
<li> ABC Rhl b 6</li>
<li> ABC Rhl l 1</li>
<li> OLasCb 32</li>
<li> ORhlB5 40</li>
<li> ApqsR 7</li>
<li> ABC Rhl m 1</li>
<li> ACE 2</li>
<li> Cl 1</li>
<li> AbaR 3</li>
<li>CEA 2</li>
</ol>

<li> Colony PCR: 5*7=35 tubes today</li>
<ol>

<li>NRhlB5 αβγ</li>
<li> NRhlB5 γαβ</li>
<li> C1BCI-pCIB5</li>
<li> BLasBCI-pCIB5</li>
<li> C1-B5</li>
<li> ACin 1</li>
<li> ACin 2</li>
</ol>

Results(+ Friday 30 tubes)


<li>Inoculation: 10 tubes all with Chl</li>
<li> C1BCI-pCIB5  9, 10</li>
<li> BLasBCI-pCIB5  13, 14</li>
<li> C1-B5  17, 19</li>
<li> ACin 2  29, 30</li>
<li> ACin 1  33, 35</li>

</html>

==2013.09.10==
<html>
<ol>

<li>Inoculation: 5 x3 tubes=15 tubes, all with Chl</li>
<ol>

<li> AbaR 3</li>
<li>Cl 1</li>
<li> ApqsR 7</li>
<li> ORhlB5 40</li>
<li> OLasCb 32</li>
</ol>

<li> Mini</li>
<ol>

<li>C1BCI-pCIB5  9, 10</li>
<li> BLasBCI-pCIB5  13, 14</li>
<li> C1-B5  17, 19</li>
<li> ACin 2  29, 30</li>
<li> ACin 1  33, 35</li>
</ol>
</ol>

</html>


==2013.09.11==
<html>
<ol>
<li> Mini: all with pSB1C3!</li>
<ol>

<li> AbaR</li>
<li> Cl</li>
<li> ApqsR</li>
<li> ORhlB5</li>
<li> OLasCb</li>
</ol>

<li> Digestion
<ol>
<li> ACin: X/P</li>
<li> C1B5: X/P  wrote wrong then cut wrong QQ</li>
<li> C1BCI pCIB5: S/P</li>
<li> BLasBCI pCIB5: S/P</li>
<li> PcA: X/P</li>
<li> PcD: X/P</li>
<li> J23119: X/P  S/P</li>
</ol>

<li> Ligation</li>
<ol>
<li> Cb-pSB1C3</li>
<li> ACin-pSb1C3</li>
<li> J23119-ACin</li>
<li> C1BCI pCIB5-PcA</li>
<li> BLasBCI pCIB5-PcD</li>
</ol>

Ligation at 16 degree O/N

<li> Transformation: retransform those 9/9 failed</li>
<ol>
<li> ABC Rhl m  Chl</li>
<li> ABC Rhl l  Chl</li>
<li> ACE  Chl</li>
<li> CEA  Chl</li>
</ol>

<li> Inoculation
<ol>
<li> NRhlB5 γαβ  1, 2  Amp</li>
<li> NRhlB5 αβγ  5, 8  Amp</li>
<li>1& 5 inoculated two, one for functional assay. </li>
<li> OLasCb x3  Chl</li>
<li> ORhlB5 x3  Chl</li>
</ol>


<li> Send plasmid to IGEM!!! </li>
<ol>

<li> AbaR</li>
<li> Cl</li>
<li> ApqsR</li>
<li> ORhlB5</li>
<li> OLasCb</li>
<li> PQSR</li>
<li> pPQS</li>
</ol>

</ol>
</html>
==2013.09.12==
<html>
<ol>
<li> Mini</li>
<ol>
<li> NRhlB5 γαβ  1, 2</li>
<li> NRhlB5 αβγ  5, 8</li>
<li> OLasCb</li>
<li> ORhlB5</li>

All with strange A260/280…, but still use 1&2

<li> Digestion</li>
<ol>
<li> NRhlB5 γαβ 1: E/P</li>
<li> NRhlB5 αβγ 8: E/P</li>
<li> C1B5 (=19): S/P</li>
<li> pPQS 2: S/P</li>
</ol>

<li> Ligation</li>
<ol>

<li> NRhlB5 γαβ-pSB1C3</li>
<li> NRhlB5 αβγ-pSB1C3</li>
<li> C1B5-PcA</li>
<li> pPQS-E1</li>
<li> J23119-ApqsR</li>
</ol>

<li> Transformation</li>
<ol>
<li> NRhlB5 γαβ-pSB1C3   Chl</li>
<li> NRhlB5 αβγ-pSB1C3   Chl</li>
<li> C1B5-PcA   Amp</li>
<li> pPQS-E1   Chl</li>
<li> J23119-ApqsR   Chl</li>
<li> Cb-pSB1C3   Chl</li>
<li> ACin-pSb1C3   Chl</li>
<li> J23119-ACin   Chl</li>
<li> C1BCI pCIB5-PcA   Amp</li>
<li> BLasBCI pCIB5-PcD   Amp</li>
</ol>

<li> Inoculation: total 10 tubes</li>
<ol>
<li> ABC Rhl m-pSb1C3  Chl</li>
<li> ABC Rhl l-pSB1C3  Chl</li>
<li> ACE-pSB1C3  Chl</li>
<li> CEA-pSB1C3  Chl</li>
<li> OLascb-pSB1C3  Chl  x2</li>
<li>ORhlB5-pSB1C3  Chl  x2</li>
<li> BLasE1PcD  Amp  x2</li>
</ol>
</ol>
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