Team:Kyoto/LabWork7

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= Lab Work Week7: Monday 12th September - Sunday 18th September =
 +
'''Monday'''<br/>
 +
 +
Luminescence:大腸菌の形質転換(Hashiya)　ハエの走行性実験②(Kusaba, Hara)
 +
 +
'''Tuesday'''<br/>
 +
 +
Luminescence:大腸菌はじめて光る。しかし光量は少ない。
 +
 +
'''Wednesday'''<br/>
 +
 +
'''Thursday'''<br/>
 +
 +
'''Friday'''<br/>
 +
 +
Digestion(Kajita)
 +
 +
PCR amplification of SAM-P20 and ChiA
 +
 +
:We performed colony direct PCRs from a ''S. albogriseolus'' colony and a ''S. avermitilis'' colony.
 +
 +
:{|class="wikitable"
 +
|+Reaction mixture
 +
!Component||Volume(&mu;l)
 +
|-
 +
|2x Buffer||25
 +
|-
 +
|2mM dNTPs||10
 +
|-
 +
|Primer 1||1.5
 +
|-
 +
|Primer 2||1.5
 +
|-
 +
|Template||X
 +
|-
 +
|KOD FX||1
 +
|-
 +
|ddH<sub>2</sub>O||up to 50
 +
|}
 +
:{|class="wikitable"
 +
|+PCR condition
 +
|Predenature||94C||2m||
 +
|-
 +
|Denature||98C||10s||rowspan="3"|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'''<br/>
 +
 +
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
 +
::1&mu;l 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/&mu;l
 +
:ChiA: 30.0 ng/&mu;l
 +
 +
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'''<br/>
 +
 +
Digestion (Kajita)
 +
 +
Purification of digested products
 +
:SAM-P20 : 10.2 ng/&mu;l
 +
:ChiA : 22.5 ng/&mu;l
 +
 +
 +
Ligation
 +
:{|class="wikitable"
 +
!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
</div>
</div>

Latest revision as of 06:06, 26 September 2011

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
ComponentVolume(μl)
2x Buffer25
2mM dNTPs10
Primer 11.5
Primer 21.5
TemplateX
KOD FX1
ddH2Oup to 50
PCR condition
Predenature94C2m
Denature98C10s30cycles
Annealing56C30s
Extension68C1m30s
We prepared two kinds of templates:
  1. Picked a colony, suspended in 50ul of water and incubated 1min at 95 degree. Added 1ul to the reaction mixture.
  2. 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:
  1. Picked a colony, suspended in 50ul of TE buffer (pH8.0) and incubated 1min at 95 degrees. Added 5ul to the reaction mixture.
  2. 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:
  1. SAM-P20 with EcoRI and SpeI
  2. 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

NameVectorInsert
1pSB1C3SAM-P20
2pSB1C3ChiA
3BBa_B0015SAM-P20
4BBa_B0015ChiA
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

Retrieved from "http://2011.igem.org/Team:Kyoto/LabWork7"