Team:Chiba/Calendar-Home/1 September 2008

From 2008.igem.org

Revision as of 07:26, 24 October 2008 by Yoshimi (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

>Home | Notebook

31 August 2008 <|> 2 September 2008

Laboratory work

Team:Input

Ptet＋RBS＋cIの、Mini Prep産物の濃度チェック。
-Ptet-cI-pMB1-Amp-の機能チェック。
-Ptet-cI-pMB1-Amp-,-PcI-GFP-p15A-Cm-をDouble Transformation(BWΔflic)
結果-->GFPが発現していた。-->-PcI-GFP-p15A-Cm-が機能していないのでは？
ターミネーターをつける、pSB1A3にのせかえる。
-Ptet-cI-pMB1-Amp-のDigestion Check

混ぜ表

	Double	Single
dH₂O	1	2
XbaI	1	1
SpeI	1	-
BSA(×10)	1	1
NEB(×10)	1	1
DNA	5	5
TOTAL	10	10

UV照射テスト

-Ptrc-LuxR-Plux-cI-colE1-Amp-,-PcI-GFP-p15a-Cm-の2plasmid(BW)
グリストをつついて2ml培養(UV⊕用と、UV⊖用の2本)
37℃,12h後UV測定(UV⊕:5.21,UV⊖:5.38)
小さなプレートにLB培地ごと流してUV照射開始。(UVは254nm,UVまでの距離は7.5cm,UV⊖のものは暗所に置いておく。)
乾かないようにポリエチレン製サランラップで蓋をした。
UV⊕はUV照射後0min,10min,30min,1h,2h,4h,6h,8に、UV⊖は0h,1h,4h,8hによく攪拌してから、20μl採取。それぞれを
10⁴,10⁵希釈し、20μlをプレートに撒いた。
37℃,12h後にコロニーを数える。

コロニー数

	UV+ 10⁴	UV+ 10⁵	UV-10⁴	UV-10⁵
0min	423	74	271	156
10min	301	51	-	-
30min	139	7	-	-
1h	89	5	1040	54
2h	5	1	-	-
4h	2	0	254	50
6h	0	0	-	-
8h	1	0	1155	106

Team:Communication

(31/8)--->Gel Check

Sample No.	1~3	4
Sample DNA	20	15
Loading Dye	4	3
TOTAL	24	18

From left;

insert-1(I9026)

insert-2(I9030)

insert-3(S03154)

vector-4(R0010)

--->Gel extract

--->zymo

insert-1(I9026) -> 7μL

insert-2(I9030) -> 7μL

insert-3(S03154) -> 7μL

vector-4(R0010) -> 15μL

--->SAP

vector-4(R0010)

--->Zymo

vector-4(R0010) -> 20μL

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6

From left;

insert-1(I9026) -> OK
insert-2(I9030) -> OK
insert-3(S03154) -> None --> Transformation [http://partsregistry.org/Part:BBa_S03154 BBa_S03154]-->

(2/9)Mini prepwith [http://partsregistry.org/Part:BBa_K084009 BBa_K084009], [http://partsregistry.org/Part:BBa_K084010 BBa_K084010]

vector-4(R0010)

--->Ligation

Sample No.	(1)	(2)	(3)	(4)	(5)
insert-1(I9026)	3	-	3	-	-
insert-2(I9030)	-	3	-	3	-
vector-4(R0010)	3	3	-	-	3
ligase	1	1	1	1	1
Buffer	1	1	1	1	1
dH₂O	2	2	5	5	5
TOTAL	10	10	10	10	10

--->Transformation

Competent cells : XL10GOLD 30μL

Transformed the following and grew on new ampicillin plates.

[http://partsregistry.org/Part:BBa_K084009 BBa_K084009(Plac+RBS+RhlI+LVA, Amp)] -> 628 colonies
[http://partsregistry.org/Part:BBa_K084010 BBa_K084010(Plac+RBS+CinI+LVA, Amp)] -> 500 colonies
insert-1(RBS+RhlI+LVA) -> 9 colonies
insert-2(RBS+CinI+LVA) -> No colonies on the plate
vector-4(Plac, Amp) -> 186 colonies

--->(2/9) Colony PCR

Transformation

Competent cells : JW1908 40μL

Transformed the following and grew on new ampicillin plates.

[http://partsregistry.org/Part:BBa_K084007 BBa_K084007(Plac+RBS+LasI)]
[http://partsregistry.org/Part:BBa_K084008 BBa_K084008(Plac+RBS+RhlI)]
[http://partsregistry.org/Part:BBa_K084010 BBa_T9002(Ptet+RBS+LuxR+GFP)]

--->(2/9)Liquid Culture

Team:Output

Ligation

vector:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] insert:[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
negative control:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010]②

Sample No.	①	②
vector	2	2
insert	3	0
Ligase Buffer	2	2
Ligase	1	1
dH₂O	3	5
TOTAL	10	10

-->R/T 2hour

Transformation

[http://partsregistry.org/Part:BBa_R0079 BBa_R0079](Las promoter)
[http://partsregistry.org/Part:BBa_R0071 BBa_R0071](RhlR promoter)
[http://partsregistry.org/Part:BBa_R0077 BBa_R0077](cinR promoter+RBS)
[http://partsregistry.org/Part:BBa_R0078 BBa_R0078](cinR promoter)
[http://partsregistry.org/Part:BBa_R0062 BBa_R0062](Lux promoter)