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Team:Kyoto/Notebook
From 2010.igem.org
LastModified: 2010-9-30
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Notebook
Tuesday, July 20 By: Wataru, Tomo, Yuki, Kazuya, Ken, Makoto
Solubilization of Antibiotics
| Ampicillin(Amp) | Kanamycin(Kan) | |
| Mix 1.0g Amp and 20ml MilliQ | Mix 0.5g Kan and 10ml MilliQ | Final concentration is 50mg/ml |
| Dispense 1.1ml of the solution into 1.5ml tubes | ||
| Store in the freezer (-20℃) | ||
Making plates for LB (Amp+) and LB (Kan+)
Transformation
| Name | Well | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|---|
| <partinfo>J23100</partinfo> | 1-18-C | 1 | 20 | 21 | LB (Amp+) | 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 (Kan+) | ● |
A vector of "pSB4K5" is Kanamycin-resistance, however, we plated it to LB plate (Amp+). 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 Y.
Culture of plates in which colonies was observed at 37℃ from 07/21 20:50 to 07/22 17:00
Making a master plate of the above plates
Retry Transformation
| Name | Well | Sample (µl) | Competent Cells (µl) | Total (µl) | Plate | Incubation | Result |
|---|---|---|---|---|---|---|---|
| <partinfo>pSB4K5</partinfo> | 1-5-G | 1 | 20 | 21 | LB (Kan+) | At 37℃ 7/21 20:50 - 7/22 16:30 | ○ |
| <partinfo>B0015</partinfo> | 1-23-L | 1 | 20 | 21 | ○ |
PCR for S-R-Rz/Rz1 and S
| No. | Water | 25mM MgSO4 | 2mM dNTPs | 10xBuffer for KOD Plus ver.2 | TemplateDNA (5ng/µl) | Primer 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 |
| 94℃ | 2min | |
| 98℃ | 10sec | 30 cycles |
| 55℃ | 30sec | |
| 68℃ | 4min | |
| 4℃ | forever |
