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Team:Kyoto/LabWork7
From 2011.igem.org
Lab Work Week7: Monday 12th September - Sunday 18th September
Monday
Luminescence:大腸菌の形質転換(Hashiya) ハエの走行性実験②(Kusaba, Hara)
Tuesday
Luminescence:大腸菌はじめて光る。しかし光量は少ない。
Wednesday
Thursday
Friday
Digestion(Kajita)
PCR amplification of SAM-P20 and ChiA
- We performed colony direct PCRs from a S. albogriseolus colony and a S. avermitilis colony.
Reaction mixture Component Volume(μl) 2x Buffer 25 2mM dNTPs 10 Primer 1 1.5 Primer 2 1.5 Template X KOD FX 1 ddH2O up to 50
PCR condition Predenature 94C 2m Denature 98C 10s 30cycles Annealing 56C 30s Extension 68C 1m30s
- We prepared two kinds of templates:
- Picked a colony, suspended in 50ul of water and incubated 1min at 95 degree. Added 1ul to the reaction mixture.
- Picked a colony and dipped in the reaction mixture.
- Gel electrophoresis indicated that the PCR amplifications were successful for a sample, ChiA, but it was a faint band. We decided to retry direct PCR of SAM-P20 and PCR of ChiA.
Saturday
Digestion(Kajita)
Retry of PCR amplification of SAM-P20 and ChiA.
- We amplified SAM-P20 by colony direct PCR and ChiA by PCR using the PCR product that performed yesterday.
- Reaction mixture: The same components and volume as before.
- PCR condition: The same PCR condition as before.
- PCR for SAM-P20
- We prepared two kinds of templates:
- Picked a colony, suspended in 50ul of TE buffer (pH8.0) and incubated 1min at 95 degrees. Added 5ul to the reaction mixture.
- Picked a colony and dipped in the reaction mixture.
- PCR for ChiA
- 1μl of PCR product was added to the reaction mixture as template.
- Gel electrophoresis indicated that the PCR amplifications were successful for all samples. However, an unexpected faint band, about 1000bp, was also observed in the sample of ChiA.
PCR purification of SAM-P20 and gel extraction of ChiA.
- SAM-P20: 43.9 ng/μl
- ChiA: 30.0 ng/μl
Restriction enzyme digestion
- We performed restriction digestions for:
- SAM-P20 with EcoRI and SpeI
- ChiA with EcoRI and SpeI
- Incubated overnight at 37 degrees.
Sunday
Digestion (Kajita)
Purification of digested products
- SAM-P20 : 10.2 ng/μl
- ChiA : 22.5 ng/μl
Ligation
Name Vector Insert 1 pSB1C3 SAM-P20 2 pSB1C3 ChiA 3 BBa_B0015 SAM-P20 4 BBa_B0015 ChiA
- Incubated overnight at 16 degrees.
PCR amplification of BBa_R0011, lactose promoter
- We done the amplification of lactose promoter. After that, we digested the product with DpnI, incubated 1 hour at 37 degrees.
- Gel electrophoresis indicated that the PCR amplification was successful, but DpnI didn't work at all. So, we done the gel extraction.
Restriction enzyme digestion for pSB4K5 with EcoRI and PstI
