Team:Chiba/jk/γ/week 2

From 2008.igem.org

>実験ログ

>出力班

Contents

WEEK 2

24,August 2008

Transformation

  • [http://partsregistry.org/Part:BBa_J63001 BBa_J63001](yeast optimized YFP)-->colony

Digestion

  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](1)①
  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2)②
  • Menbren Venus YFP③
Sample No.
DNA tamplate 333
Buffer3 111
dH2O 666
EcoRⅠ 0.50.50.5
TOTAL 101010


-->37℃ 30min


Sample No. ①(afetr digestion)②(after digestion)③(afetr digestion)
DNA tamplate 101010
loading Dye 222
TOTAL 121212


Sample No.
DNA tamplate 222
loading Dye 111
dH2O 333
TOTAL 666

-->135V 30min


25,August,2008

PCR

  • [http://partsregistry.org/Part:BBa_E2030 BBa_E2030]①
  • [http://partsregistry.org/Part:BBa_J33202 BBa_J33202]②
  • BL21③
  • [http://partsregistry.org/Part:BBa_J52008 BBa_J52008]④
  • [http://partsregistry.org/Part:BBa_I712019 BBa_I712019]⑤
>
Sample No.
DNA tamplate 11111
FW primer 5(Venus_YFP_fwd)5(LacZ_fwd)5(LacZ_fwd)5(rLUC_fwd)5(fLuc_fwd)
VR primer 5(VR)5(LacZ_rev)5(LacZ_rev)5(VR)5(VR)
dNTPmix 1010101010
Thermo pol buffer 1010101010
DNA pol(VENT) 11111
dH2O 6868686868
TOTAL 100100100100100

-->Gel Check


>
Sample No.
DNA tamplate 11111
loading Dye 11111
dH2O 44444
TOTAL 66666

Mini prep

  • Venus
  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]


Transformation

  • Venus
  • PUC19
  • pGFPUV
  • pGEX Venus YFP
  • Membren Venus YFP

PCR

  • [http://partsregistry.org/Part:BBa_J63001 BBa_J63001]①
Sample No.
DNA tamplate 1
FW primer(Venus) 5
VR primer 5
dNTPmix 10
Thermo pol buffer 10
DNA pol(VENT) 1
dH2O 68
TOTAL 100

26,August,2008

Gel Check

  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
  • [http://partsregistry.org/Part:BBa_J63001 BBa_J63001]
>
Sample No.
DNA tamplate 11111
Loading Dye 11111
dH2O 44444
TOTAL 66666

Digestion

  • vector:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
Sample No. [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
DNA tamplate 100
SpeⅠ 2
PstⅠ 2
BSA(x10) 13
Buffer2 13
TOTAL 130
  • insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008],[http://partsregistry.org/Part:BBa_E2030 BBa_E2030]
Sample No. [http://partsregistry.org/Part:BBa_J52008 BBa_J52008][http://partsregistry.org/Part:BBa_E2030 BBa_E2030]
DNA tamplate 3030
XbaⅠ 11
PstⅠ 11
DpnⅠ 11
Buffer3 44
BSA(x10) 44
TOTAL 4040


-->37℃ 3hour
-->SAP
-->37℃ 30min
-->65℃ 15min
-->Zymo Clean

Ligation

  • vector:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
  • vector:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620] insert:[http://partsregistry.org/Part:BBa_E2030 BBa_E2030]②
  • Negative control:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620]③
Sample No.
vector 555
inser 107.50
Ligase Buffer 444
Ligase 111
dH2O 02.510
TOTAL 202020

-->RT 2hour

-->Transformation(XL10G)

-->37℃ over night

-->colony PCR

-->Mini prep

28,August,2008

Time Responce(liquid)


29,August,2008

TIME RESPONCE(solid)


30,August,2008

Transformation

  • mcherry

PCR

  • [http://partsregistry.org/Part:BBa_J52008 BBa_J52008](rLuc)
Sample No. [http://partsregistry.org/Part:BBa_J52008 BBa_J52008]
DNA tamplate 1
rLuc_fwd 2.5
Rev primer 2.5
Thermo pol Buffer 5
dNTPmix 5
Vent pol 0.5
dH2O 34
TOTAL 50



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