Team:HokkaidoU Japan/Notebook/asB0034/asE1010 1A3 Construction

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Notebook

Lab Documents

Anti-Sense E1010 on 1A3 Construction

Start
PCR
- Wed Sep 24
R0010-stem-asE1010-stem-B0015(on pSB1C3) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R(insert) Tm:72.6℃

KOD Fx Neo
Digestion
- Thu Sep 25
Cut vector with EcoRI, PstI (using 10×H buffer)

Cut insert with EcoRI, PstI (using 10×M buffer)
Dephosphorylation
- Fri Sep 26
Antactic phosphorylate pSB1A3 (using 10×Antarctic phosphatase buffer) 37°C 35min 65℃ 10min
Ligation
- Fri Sep 26
Ligate pSB1A3 with R0010-stem-asE1010-stem-B0015
Transformation
- Fri Sep 26
R0010-stem-asE1010-stem-B0015(on pSB1A3)

5.0µL to DH5α
Colony PCR
- Sat Sep 27
R0010-stem-asE1010-stem-B0015(on pSB1A3) EcoI_XbaI_F Tm:74.5℃ & PstI_SpeI_R Tm:74.6℃

Kapa-Taq
Culture
- Sat Sep 27
R0010-stem-asE1010-stem-B0015(on pSB1A3)
Mini-Prep
- Sun Sep 28
R0010-stem-asE1010-stem-B0015(on pSB1A3)
Complete!