Team:Chiba/Calendar-Home/1 September 2008

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31 August 2008 <|> 2 September 2008

Laboratory work

Team:Input

Gel Check

Ptet＋RBS＋cI

Transformation(Double Transformation)

-Ptet-cI-pMB1-Amp-,-PcI-GFP-p15A-Cm-(JW1908Δflic)

Digestion test

-Ptet-cI-pMB1-Amp-

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

UV irradiation test

two plasmids from (JW1908)
- -Ptrc-LuxR-Plux-cI-colE1-Amp-
- -PcI-GFP-p15a-Cm-

Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium for 12 hours at 37 degrees.

OD:UV⊕:5.21,UV⊖(negative control):5.38

moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.Put the negartive control in a dark place.
covered the plates with polyethylene wrap.
after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h),we agitate the culture and took 20μl from each other
diluted each cultures with LB-ampicillin medium 10⁴-fold and 10⁵-fold (volume/volume).
incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
counted the CFU(determined viable cell count).

Viable cell count

	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	24μl	18μl

From left;

insert-1(BBa_I9026)

insert-2(BBa_I9030)

insert-3(BBa_S03154)

vector-4(BBa_R0010)

--->Gel extract

--->zymo

insert-1(BBa_I9026) -> 7μL

insert-2(BBa_I9030) -> 7μL

insert-3(BBa_S03154) -> 7μL

vector-4(BBa_R0010) -> 15μL

--->SAP

vector-4(BBa_R0010)

--->Zymo

vector-4(BBa_R0010) -> 20μL

--->Gel Check

Sample DNA	1
Loading Dye	1
dH₂O	4
TOTAL	6μl

From left;

insert-1(BBa_I9026) -> OK
insert-2(BBa_I9030) -> OK
insert-3(BBa_S03154) -> None --> Transformation BBa_S03154-->

(2/9)Mini prepwith BBa_K084009, BBa_K084010

vector-4(BBa_R0010)

--->Ligation

Sample No.	(1)	(2)	(3)	(4)	(5)
insert-1(BBa_I9026)	3	-	3	-	-
insert-2(BBa_I9030)	-	3	-	3	-
vector-4(BBa_R0010)	3	3	-	-	3
ligase	1	1	1	1	1
Buffer	1	1	1	1	1
dH₂O	2	2	5	5	5
TOTAL	10μl	10μl	10μl	10μl	10μl

--->Transformation

Competent cells : XL10GOLD 30μL

Transformed the following and grew on new ampicillin plates.

BBa_K084009(Plac+RBS+RhlI+LVA, Amp) -> 628 colonies
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.

--->(2/9)Liquid Culture

Team:Output

Ligation

vector:BBa_R0010 insert:BBa_J52008(1)
negative control:BBa_R0010(2)

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

-->R/T 2hour

Transformation