Team:Chiba/jk/γ/week 2

From 2008.igem.org

>実験ログ

>出力班

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.	①	②	③
DNA tamplate	3	3	3
Buffer3	1	1	1
dH₂O	6	6	6
EcoRⅠ	0.5	0.5	0.5
TOTAL	10	10	10

-->37℃ 30min

Sample No.	①(afetr digestion)	②(after digestion)	③(afetr digestion)
DNA tamplate	10	10	10
loading Dye	2	2	2
TOTAL	12	12	12

Sample No.	①	②	③
DNA tamplate	2	2	2
loading Dye	1	1	1
dH₂O	3	3	3
TOTAL	6	6	6

-->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.	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
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	10	10	10	10	10
Thermo pol buffer	10	10	10	10	10
DNA pol(VENT)	1	1	1	1	1
dH₂O	68	68	68	68	68
TOTAL	100	100	100	100	100

-->Gel Check

>

Sample No.	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
loading Dye	1	1	1	1	1
dH₂O	4	4	4	4	4
TOTAL	6	6	6	6	6

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.	①
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.	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
Loading Dye	1	1	1	1	1
dH₂O	4	4	4	4	4
TOTAL	6	6	6	6	6

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	30	30
XbaⅠ	1	1
PstⅠ	1	1
DpnⅠ	1	1
Buffer3	4	4
BSA(x10)	4	4
TOTAL	40	40

-->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.	①	②	③
vector	5	5	5
inser	10	7.5	0
Ligase Buffer	4	4	4
Ligase	1	1	1
dH₂O	0	2.5	10
TOTAL	20	20	20

-->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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Team:Chiba/jk/γ/week 2

From 2008.igem.org

Contents

WEEK 2

24,August 2008

25,August,2008

26,August,2008

28,August,2008

29,August,2008

30,August,2008