Team:Chiba/Calendar-Home/26 August 2008

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25 August 2008 <|> 27 August 2008

Laboratory work

Team:Input

no work

Team:Communication

(8/25)--->Gel Check

Sample No. 1~3 Sample DNA(μL) 30 Loading Dye(μL) 6 TOTAL(μL) 36 1, [http://partsregistry.org/Part:BBa_C0178 BBa_C0178] Insert:609bp -> OK

2, [http://partsregistry.org/Part:BBa_C0170 BBa_C0170] Insert:609bp -> OK

3, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) --> OK

--->Gel Extract

--->Zymo Clean

eluted with following amount of elution buffer

1. 5μl
2. 5μl
3. 27μl

--->Gel Check

Sample No. 1~3 Sample DNA(μL) 2 Loading Dye(μL) 2 H2O(μL) 8 TOTAL(μL) 12 From left; insert:[http://partsregistry.org/Part:BBa_C0178 BBa_C0178] --> OK insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] --> OK? vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500] --> OK

--->SAP

Sample No.	3
Sample DNA(μL)	26
SAP(μL)	1
SAP Buffer(μL)	3
TOTAL(μL)	30

--->Zymo Clean

Sample No.3 -->9μl

--->Gel Check

Sample No.3 1 Loading Dye(μL) 1 dH2O(μL) 4 TOTAL(μL) 6 Sample No.3 -> OK ･･･30ng?

--->Ligation

Sample No.	(1)	(2)	(3)	(4)	(5)
insert(1)[http://partsregistry.org/Part:BBa_C0178 BBa_C0178](μL)	1	-	-	1	-
insert(2)[http://partsregistry.org/Part:BBa_C0170 BBa_C0170](μL)	-	1	-	-	1
vector(3)[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](μL)	-	-	2	2	2
ligase(μL)	1	1	1	1	1
Buffer(μL)	2	2	2	2	2
dH2O(μL)	6	6	5	4	4
TOTAL(μL)	10	10	10	10	10

--->Transformation

1. insert(1)[http://partsregistry.org/Part:BBa_C0178 BBa_C0178]
2. insert(2)[http://partsregistry.org/Part:BBa_C0170 BBa_C0170]
3. vector(3)[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
4. insert(1)[http://partsregistry.org/Part:BBa_C0178 BBa_C0178]+vector(3)[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
5. insert(2)[http://partsregistry.org/Part:BBa_C0170 BBa_C0170]+vector(3)[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]

Team:Output

Gel Check

• [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](1)
• [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2)
• [http://partsregistry.org/Part:BBa_J63001 BBa_J63001](3)

Sample No.	1	2	3
DNA tamplate	1	1	1
Loading Dye	1	1	1
dH2O	4	4	4
TOTAL	6μl	6μl	6μl

Digestion

• vector:[http://partsregistry.org/Part:BBa_F2620 BBa_F2620](1)

Sample No.	1
DNA tamplate	100
SpeI	2
PstI	2
BSA(x10)	13
Buffer2	13
TOTAL	130μl

• insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008](1)

[http://partsregistry.org/Part:BBa_E2030 BBa_E2030](2)

Sample No.	1	2
DNA tamplate	30	30
XbaI	1	1
PstI	1	1
DpnI	1	1
Buffer3	4	4
BSA(x10)	4	4
TOTAL	40μl	40μl

-->37°C　3hour
-->SAP
-->37°C 30min
-->65°C 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	20μl	20μl	20μl

-->RT 2hour

-->Transformation(XL10G)

-->37°C over night

-->colony PCR

-->Mini prep

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