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

5μl
5μl
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

insert(1)[http://partsregistry.org/Part:BBa_C0178 BBa_C0178]
insert(2)[http://partsregistry.org/Part:BBa_C0170 BBa_C0170]
vector(3)[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
insert(1)[http://partsregistry.org/Part:BBa_C0178 BBa_C0178]+vector(3)[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
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]
[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	6μl	6μl	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