Tuesday, July 20
| By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
|
| (1) Solubilization of antibiotics.
| For Ampicillin(Amp): add 1.0g Amp to 20ml MilliQ (Final concentration is 50mg/ml).
| For Kanamycin(Kan): add 0.5g Kan to 10ml MilliQ (Final concentration is 50mg/ml).
| Dispense 1.1ml of the solution into 1.5ml tubes.
| Store in the freezer (-20℃).
|
| (2) Making plates for LB (Amp+) and LB (Kan+).
|
(3) Transformation of iGEM Parts.
| Name | Well | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result
|
| <partinfo>J23100</partinfo> | 1-18-C | 1 | 20 | 21 | LB (Ampicillin+) | At 37℃ 7/20 20:50 - 7/21 17:00 | ○
|
| <partinfo>J23105</partinfo> | 1-18-M | 1 | 20 | 21 | ○
|
| <partinfo>J23116</partinfo> | 1-20-M | 1 | 20 | 21 | ○
|
| <partinfo>R0011</partinfo> | 1-6-G | 1 | 20 | 21 | ○
|
| <partinfo>E0840</partinfo> | 1-12-O | 1 | 20 | 21 | ○
|
| <partinfo>J06702</partinfo> | 2-8-E | 1 | 20 | 21 | ○
|
| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | ×
|
| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | LB (Kanamycin+) | ×
|
| *1 "1-18-C" means well 18C in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
| Discussion
| A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Ampicillin+).
| And We didn't pre-culture "B0015" despite its vector is Kanamycin-resistance.
| So, it was predicted that we will fail the transformation of "pSB4K5" and "B0015".
|
Wednesday, July 21
- By: Wataru, Ken, Makoto, Takuya Yamamoto
- Category: Transformation, PCR, Lysis Cassette
|
| (1) Cultured plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.
|
| (2) Made a master plate of the above plates.
|
(3) Retried Transformation of iGEM Parts.
| Name | Well*1 | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result
|
| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | LB (Kanamycin+) | At 37℃ 7/21 20:50 - 7/22 16:30 | ○
|
| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | ○
|
- *1 "1-5-G" means well 5G in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
|
(4) PCR for S-R-Rz/Rz1 and S
- Dilute λDNA (0.5µg/µl) 100 times with MilliQ. The final concentration of template λDNA was 5ng/µl.
| No. | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | TemplateDNA (5ng/µl) | Primer S-R-Rz/Rz1 Forward (10µM) | Primer S-R-Rz/Rz1 Reverse (10µM) | Primer S Reverse (10µM) | KOD Plus ver.2 | Total
|
| 1 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
|
| 2 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
|
| 3 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
|
| 4 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
|
| 5 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
|
| 6 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
|
| 7 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
|
| 8 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
|
- Forward Primer of S-R-Rz/Rz1 and S is common.
- PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.
|
Friday 23, July
- By: Wataru, Tomo, Makoto
- Category:
|
(1) Miniprep of iGEM Parts.
| Name | Concentration(ng/µl)
|
| <partinfo>pSB4K5</partinfo> | 79.2
|
| <partinfo>B0015</partinfo> | -
|
- Discussion
- We lost <partinfo>B0015</partinfo> by our mistake.
- The concentration of <partinfo>pSB4K5</partinfo> is high, so this condition of shaking incubation is moderate.
|
(2) Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.
| Sample | Concentration (ng/µl) | New Name |
|
| 1 | 18.6 | -
|
| 3 | 77.6 | S1
|
| 5 | 33.6 | -
|
| 7 | 65.4 | S2
|
- Discussion
- The concentration of sample number 1 and 5, the PCR products of S-R-Rz/Rz1, is week, so we desided to retry PCR.
|
(3) Retry of PCR of S-R-Rz/Rz1.
| Sample | Water | 25mmol/l MgSO4 | 2mmol/l dNTPs | 10×Buffer for KOD plus ver.2 | Template DNA (5ng/µl) | Primer S-R-Rz/Rz1 Forward (10µmol/l) | Primer S-R-Rz/Rz1 Reverse (10µmol/l) | KOD plus ver.2 | Total
|
| 1 | 28µl | 3 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
|
| 2 | 28 | 3 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
|
| 3 | 26.5 | 4.5 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
|
| 4 | 26.5 | 4.5 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
|
| 5 | 25 | 6 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
|
| 6 | 25 | 6 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
|
- PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.
|
(4) Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.
| Sample | 10xBuffer | BSA | Enzyme | MilliQ | Total | Incubation
|
| 1 | 5µl | 1 | EcoRI 0.1 | 3.6 | 10 | At 37℃ 7/23 18:00 - 7/23 18:30
|
| 2 | 5 | 1 | XbaI 0.1 | 3.6 | 10
|
| 3 | 5 | 1 | SpeI 0.1 | 3.6 | 10
|
| 4 | 5 | 1 | PstI 0.1 | 3.6 | 10
|
| 5 | 5 | 1 | - | 3.7 | 10
|
|
(5) Electrophoresis of above sample for 35min.
- Discussion
- Comparison to sample 5(control, circular DNA), the bands of sample 1, 2, 3, 4 was shifted. The DNA of sample 1, 2, 3, 4 was linearized by Restriction enzymes. So, our restriction enzymes work correctly.
|
(6) To insert S gene to GFP, we digested the PCR products of S gene by EcoRi and SpeI, and GFP by EcoRl and XbaI.
| Sample | 10×Buffer | Enzyme 1 | Enzyme 2 | MilliQ | Total | Incubation
|
| S1 | 11µl | 5 | EcoRI 0.2 | SpeI 0.2 | 33.6 | 50 | At 37℃ for 2h
|
| S2 | 11 | 5 | EcoRI 0.2 | SpeI 0.2 | 33.6 | 50
|
| <partinfo>E0840</partinfo>(GFP) | 45 | 5 | EcoRI 0.2 | XbaI 0.2 | 0 | 50
|
- After PCR purification, evaporated them and diluted 3ul.
|
Ligated over night
| Sample | Vector | Insert | Ligation High | Total
|
| S-GFP1 | <partinfo>E0840</partinfo> 0.5µl | S1 0.5 | 1 | 2
|
| S-GFP2 | <partinfo>E0840</partinfo> 0.5 | S2 0.5 | 1 | 2
|
|
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