Team:Kyoto/Notebook

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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 MilliQMix 0.5g Kan and 10ml MilliQFinal 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

NameWellSample (µl)Competent Cells (µl)Total (µl)PlateIncubationResult
<partinfo>J23100</partinfo>1-18-C12021LB (Amp+)At 37℃, 7/20 20:50 - 7/21 17:00
<partinfo>J23105</partinfo>1-18-M12021
<partinfo>J23116</partinfo>1-20-M12021
<partinfo>R0011</partinfo>1-6-G12021
<partinfo>E0840</partinfo>1-12-O12021
<partinfo>J06702</partinfo>2-8-E12021
<partinfo>pSB4K5</partinfo>1-5-G12021
<partinfo>B0015</partinfo>1-23-L12021LB (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||rowspan="2"|LB (Kan+)||rowspan="2"|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.Water25mM MgSO42mM dNTPs10xBuffer for KOD Plus ver.2TemplateDNA (5ng/µl)Primer Forward (10µM)Primer S-R-Rz/Rz1 Reverse (10µM)Primer S Reverse (10µM)KOD Plus ver.2Total
128µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
228µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
328µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
428µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
528µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
628µl3µl5µl5µl5µl1.5µl1.5µl-1µl50µl
728µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
828µl3µl5µl5µl5µl1.5µl-1.5µl1µl50µl
PCR condition
94℃2min
98℃10sec30 cycles
55℃30sec
68℃4min
4℃forever