2013.9.2-9.7

2013.09.03

1. Sequencing: send 18 samples includes:
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.
2. Ligation
1. BLas-Cg (=BLas-E1)
2. BLas-Cm (=BLas-B5)
3. BLas-Cb

2013.09.04

1. Still no competent cells.
2. Check: total 24 samples
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
3. Mini
1. Pc18C-ACin 3,4
2. Pc18A-RBS-CinR 2,3
3. BCinBCI 3,4
4. BLasBCI 2,3
5. pCIB5 3
4. Digestion
1. BLas(5): E/S
2. E1-PcD(4): E/X
3. E1(1): X/P
4. ApqsR(7): X/P
5. Ligation
1. Blas-[E1-PcD]
2. pCI-E1
3. Pc-[RBS-pqsR-tt]

2013.09.05

One day more. Another day, another destiny. This never ending road of cloning~

1. Digestion
1. pCIB5(3): X/P
2. pCIB5(3): E/S
3. C1BCI(29): S/P
4. BLasBCI(2): S/P
5. B0015 8/22: E/X
6. Pc18C ACin(4): E/S
7. Pc18A-RBS-CinR(2): E/S
Since we forgot which was B0015 when gel electrophoresis > <, we use 2 B0015 when ligation.
2. Ligation
1. PcAC1BCI-pCIB5 (=NRhlB5 αβγ)
2. pCI-B5-PcAC1BCI (=NRhlB5 γαβ)
3. C1BCI-pCIB5
4. BLasBCI-pCIB5
5. Pc18C ACin-B0015 8/22 (=18CACin)
6. Pc18A ACin-B0015 8/22 (=18A ACin)
7. C1-B5
8. [RBS-CinR]-B0015 8/22 (=ACin 1)
9. [RBS-CinR]-B0015
Ligation at 16 degree overnight
3. Transformation: total 7 plates
1. BLas-[E1-PcD] Amp
2. pCI-E1 Amp
3. PcApqsR (=Pc-RBS-pqsr-tt) Amp Chl
4. BLas-Cg Amp
5. BLas-Cm Amp
6. BLas-Cb Amp
4. Sequencing: 13 samples include:
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
5. NTU-Taiwan visited us~~~
6. We have new competent cells!

2013.09.06

I dreamed a dream when time gone by, when hope was high and things worked (seemed) smoothly

1. Go to Center of Genomic Medicine to have a short presentation and course.
2. Colony PCR: 30 tubes
1. BLas-[E1-PcD]
2. pCI-E1
3. PcApqsR (=Pc-RBS-pqsr-tt)
4. BLas-Cg
5. BLas-Cm
6. BLas-Cb
Used wrong program so redid it. Run gel electrophoresis next week.
3. Transformation: 9 plates
1. NRhlB5 αβγ Amp
2. NRhlB5 γαβ Amp
3. C1BCI-pCIB5 Amp
4. BLasBCI-pCIB5 Amp
5. Pc18C ACin Amp
6. Pc18A ACin Chl
7. C1-B5 Amp
8. ACin 1 Amp
9. ACin 2 Amp
Transform at room temperature over weekend.

2013.09.09-14

2013.09.09

Crying alone is not allowed, not in my castle on the cloud.

1. Digestion: all use X/P to cut, in order to change backbone to pSB1C3
1. OB5Rhl 1
2. OCbLas 5
3. ABC Rhl b 6
4. ABC Rhl l 1
5. OLasCb 32
6. ORhlB5 40
7. ApqsR 7
8. ABC Rhl m 1
9. ACE 2
10. Cb 3
11. Cl 1
12. AbaR 3
13. CEA 2
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.
2. Ligation
1. 1~13 except Cb, total 12 samples.
2. Since we forgot to cut pSB1C3 at X/P, we were a little delay.
3. Transformation (at CGM): 12 plates, all with Chl. Use half ligation products with 50ul competent cell. Leave half ligation O/N at 4 degree.
1. OB5Rhl 1
2. OCbLas 5
3. ABC Rhl b 6
4. ABC Rhl l 1
5. OLasCb 32
6. ORhlB5 40
7. ApqsR 7
8. ABC Rhl m 1
9. ACE 2
10. Cl 1
11. AbaR 3
12. CEA 2
4. Colony PCR: 5*7=35 tubes today
1. NRhlB5 αβγ
2. NRhlB5 γαβ
3. C1BCI-pCIB5
4. BLasBCI-pCIB5
5. C1-B5
6. ACin 1
7. ACin 2
Results(+ Friday 30 tubes)
5. Inoculation: 10 tubes all with Chl
1. C1BCI-pCIB5 9, 10
2. BLasBCI-pCIB5 13, 14
3. C1-B5 17, 19
4. ACin 2 29, 30
5. ACin 1 33, 35

2013.09.10

1. Inoculation: 5 x3 tubes=15 tubes, all with Chl
1. AbaR 3
2. Cl 1
3. ApqsR 7
4. ORhlB5 40
5. OLasCb 32
2. Mini
1. C1BCI-pCIB5 9, 10
2. BLasBCI-pCIB5 13, 14
3. C1-B5 17, 19
4. ACin 2 29, 30
5. ACin 1 33, 35

2013.09.11

1. Mini: all with pSB1C3!
1. AbaR
2. Cl
3. ApqsR
4. ORhlB5
5. OLasCb
2. Digestion
   1. ACin: X/P
   2. C1B5: X/P wrote wrong then cut wrong QQ
   3. C1BCI pCIB5: S/P
   4. BLasBCI pCIB5: S/P
   5. PcA: X/P
   6. PcD: X/P
   7. J23119: X/P S/P
3. Ligation
1. Cb-pSB1C3
2. ACin-pSb1C3
3. J23119-ACin
4. C1BCI pCIB5-PcA
5. BLasBCI pCIB5-PcD
Ligation at 16 degree O/N
4. Transformation: retransform those 9/9 failed
1. ABC Rhl m Chl
2. ABC Rhl l Chl
3. ACE Chl
4. CEA Chl
5. Inoculation
   1. NRhlB5 γαβ 1, 2 Amp
   2. NRhlB5 αβγ 5, 8 Amp
   3. 1& 5 inoculated two, one for functional assay.
   4. OLasCb x3 Chl
   5. ORhlB5 x3 Chl
6. Send plasmid to IGEM!!!
1. AbaR
2. Cl
3. ApqsR
4. ORhlB5
5. OLasCb
6. PQSR
7. pPQS

2013.09.12

1. Mini
1. NRhlB5 γαβ 1, 2
2. NRhlB5 αβγ 5, 8
3. OLasCb
4. ORhlB5
All with strange A260/280…, but still use 1&2
5. Digestion
1. NRhlB5 γαβ 1: E/P
2. NRhlB5 αβγ 8: E/P
3. C1B5 (=19): S/P
4. pPQS 2: S/P
6. Ligation
1. NRhlB5 γαβ-pSB1C3
2. NRhlB5 αβγ-pSB1C3
3. C1B5-PcA
4. pPQS-E1
5. J23119-ApqsR
7. Transformation
1. NRhlB5 γαβ-pSB1C3 Chl
2. NRhlB5 αβγ-pSB1C3 Chl
3. C1B5-PcA Amp
4. pPQS-E1 Chl
5. J23119-ApqsR Chl
6. Cb-pSB1C3 Chl
7. ACin-pSb1C3 Chl
8. J23119-ACin Chl
9. C1BCI pCIB5-PcA Amp
10. BLasBCI pCIB5-PcD Amp
8. Inoculation: total 10 tubes
1. ABC Rhl m-pSb1C3 Chl
2. ABC Rhl l-pSB1C3 Chl
3. ACE-pSB1C3 Chl
4. CEA-pSB1C3 Chl
5. OLascb-pSB1C3 Chl x2
6. ORhlB5-pSB1C3 Chl x2
7. BLasE1PcD Amp x2