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Team:Kyoto/Notebook
From 2010.igem.org
LastModified: 2010-8-25
Contents |
Index
Notebook
001: Preparation of iGEM Parts
- By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
- Date: 07/20
- Category: Antibiotic, Parts, Transformation
- Protocol: Use BioBrick Parts
Solubilization fo antibiotics
- 1. Make two mixtures as the following.
- 1g Ampicillin and 20ml MilliQ (Final concentration 50mg/ml).
- 0.5g Ampicillin and 10ml MilliQ (Final concentration 50mg/ml).
- 2. Dispense 1.1ml to 1.5ml tubes.
- 3. Keep in -20℃ Freezer.
Make LB plate
Make plates for LB (Ampicillin+) and LB (Kanamycin+).
Transformation of iGEM Parts
| Name | Well*1 | 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 001
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".
002:
- By: Wataru, Ken, Makoto, Takuya Yamamoto
- Date: 07/21
- Category: Culture, MasterPlate, Transformation, Parts, PCR, LysisCassette
- Protocol: Use BioBrick Parts, PCR
Result of 001
| Name | Colony | Next Step |
|---|---|---|
| <partinfo>J23100</partinfo> | Many | Make a Master Plate, Miniprep |
| <partinfo>J23105</partinfo> | Many | |
| <partinfo>J23116</partinfo> | Many | |
| <partinfo>R0011</partinfo> | Many | |
| <partinfo>E0840</partinfo> | Many | |
| <partinfo>J06702</partinfo> | Many | |
| <partinfo>pSB4K5</partinfo> | No | Retry Transformation |
| <partinfo>B0015</partinfo> | No |
Make a Master Plate
Retry Transforamtion of iGEM Parts
| Name | Well*1 | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | |
| <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.
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.
| 94℃ | 2min | x1 |
| 98℃ | 10sec | x30 cycle |
| 55℃ | 30sec | |
| 68℃ | 1min | |
| 4℃ | forever |
