Tuesday, July 20
- By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
- Category: Antibiotic, Transformation
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(1) Solubilized of antibiotics, Ampicillin and Kanamycin as the following.
- 1. Mix the following (final concentration is 50mg/ml).
- For Ampicillin: 1g Ampicillin and 20ml MilliQ.
- For Kanamycin: 0.5g Kanamycin and 10ml MilliQ.
- 2. Dispense 1.1ml of the mixture into 1.5ml tubes.
- 3. Store in the freezer (-20℃).
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| (2) Made plates for LB (Ampicillin+) and LB (Kanamycin+).
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(3) Transformed iGEM Parts.
| Name | Well*1 | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result
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| <partinfo>J23100</partinfo> | 1-18-C | 1 | 20 | 21 | LB (Ampicillin+) | At 37℃ 7/20 20:50 - 7/21 17:00 | ○
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| <partinfo>J23105</partinfo> | 1-18-M | 1 | 20 | 21 | ○
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| <partinfo>J23116</partinfo> | 1-20-M | 1 | 20 | 21 | ○
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| <partinfo>R0011</partinfo> | 1-6-G | 1 | 20 | 21 | ○
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| <partinfo>E0840</partinfo> | 1-12-O | 1 | 20 | 21 | ○
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| <partinfo>J06702</partinfo> | 2-8-E | 1 | 20 | 21 | ○
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| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | ×
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| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | LB (Kanamycin+) | ×
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- *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".
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Wednesday, July 21
- By: Wataru, Ken, Makoto, Takuya Yamamoto
- Category: Transformation, PCR, Lysis Cassette
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| Cultured plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00.
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| Made a master plate of the above plates.
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Retried Transformation of iGEM Parts.
| Name | Well*1 | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result
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| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | LB (Kanamycin+) | At 37℃ 7/21 20:50 - 7/22 16:30 | ○
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| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | ○
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- *1 "1-5-G" means well 5G in [http://partsregistry.org/Help:Spring_2010_DNA_distribution Spring 2010 DNA Distribution Kit] Plate 1.
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PCR 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
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| 1 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
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| 2 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
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| 3 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
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| 4 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
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| 5 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
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| 6 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | - | 1µl | 50µl
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| 7 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
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| 8 | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | - | 1.5µl | 1µl | 50µl
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- 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.
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Friday 23, July
- By: Wataru, Tomo, Makoto
- Category:
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1. Miniprep of iGEM Parts.
| Name | Concentration(ng/µl)
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| <partinfo>pSB4K5</partinfo> | 79.2
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| <partinfo>B0015</partinfo> | -
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- 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.
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2. Picked up 1, 3, 5, 7 of the products of PCR, and purified by PCR-purification.
| Sample | Concentration (ng/µl) | New Name |
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| 1 | 18.6 | -
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| 3 | 77.6 | S-1
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| 5 | 33.6 | -
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| 7 | 65.4 | S-2
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- Discussion
- The concentration of sample number 1 and 5, the PCR products of S-R-Rz, is week, so we desided to retry PCR.
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3. Retry of PCR of S-R-Rz/Rz1.
| Sample | Water | 25mM MgSO4 | 2mM dNTPs | 10×Buffer for KOD plus ver.2 | Template DNA (5ng/µl) | Primer S-R-Rz/Rz1 Forward (10µM) | Primer S-R-Rz/Rz1 Reverse (10µM) | KOD plus ver.2 | Total
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| 3a | 28µl | 3µl | 5µl | 5µl | 5µl | 1.5µl | 1.5µl | 1µl | 50µl
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| 3b | 28 | 3 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
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| 4.5a | 26.5 | 4.5 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
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| 4.5b | 26.5 | 4.5 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
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| 6a | 25 | 6 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
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| 6b | 25 | 6 | 5 | 5 | 5 | 1.5 | 1.5 | 1 | 50
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- PCR condition : 94℃ x 2min, (98℃ x 10sec, 55℃ x 30sec, 68℃ x 1min) x 30cycles, 4℃ forever.
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4. Digested <partinfo>J06702</partinfo> by EcoRI, XbaI, SpeI, PstI to check function of our Restriction enzymes.
| Sample | 10xBuffer | BSA | Enzyme | MilliQ | Total | Incubation
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| 1 | 5µl | 1 | EcoRI 0.1 | 3.6 | 10 | At 37℃ 7/23 18:00 - 7/23 18:30
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| 2 | 5 | 1 | XbaI 0.1 | 3.6 | 10
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| 3 | 5 | 1 | SpeI 0.1 | 3.6 | 10
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| 4 | 5 | 1 | PstI 0.1 | 3.6 | 10
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| 5 | 5 | 1 | - | 3.7 | 10
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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.
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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
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| S-1 | 11µl | 5 | EcoRI 0.2 | SpeI 0.2 | 33.6 | 50 | At 37℃ for 2h
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| S-2 | 11 | 5 | EcoRI 0.2 | SpeI 0.2 | 33.6 | 50
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| <partinfo>E0840</partinfo>(GFP) | 45 | 5 | EcoRI 0.2 | XbaI 0.2 | 0 | 50
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- After PCR purification, evaporated them and diluted 3ul.
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Ligated over night
| Sample | Vector | Insert | Ligation High | Total
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| S-GFP-1 | <partinfo>E0840</partinfo> 0.5µl | S-1 0.5 | 1 | 2
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| S-GFP-2 | <partinfo>E0840</partinfo> 0.5 | S-2 0.5 | 1 | 2
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