Team:Chiba/Calendar-Home/21 August 2008

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20 August 2008 <|> 22 August 2008

Laboratory work

Team:Input

(-->20/8)

Inoculated transformants for 12 hours in LB 2mL containing Ampicillin.

BBa_R0051(2007)
BBa_R0051(2006)
BBa_J06650(2007)
BBa_J06650(2006)
BBa_J22136(2007)
BBa_J22141(2007)

BBa_J22141:After inoculated for 11 hours, added 20μl IPTG and cultured one hour.

UV irradiation test (plate phase)
two plasmids from(JW1908)
- (Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-
- (Reporter)-PcI-GFP-p15a-Cm-

Inoculated (Reporter) culture from glycerol stock(-80°C freezer) in 2mL LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL mediumfor 12h, at 37&degC.
We diluted 10⁵-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
We cultured for 12h at 37°C.
We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
UVを照射して9h後、21h後にそれぞれ両方のプレートに次の同じ操作をする。
両方のプレートのコロニーをつついてそれぞれ(LB-Amp,Cm),(LB-Amp,Cm,AHL100nM)2ml培養(37°C,12h)する。
We diluted 10⁵-fold, and plated 20ul of the

resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.

Colony count

	no AHL	AHL100nM
9h	606	453
12h	146	151

Colonies of no AHL plate were AHLが入っていないほうのコロニーはいづれも光っていた。→機能チェックはOK

Team:Communication

(20/8)--->PCR

BBa_C0070(2007)
BBa_C0070(2006)
BBa_C0076(2007)
BBa_C0078(2007)
BBa_C0078(2006)

DNA Template	1
dNTP mix	10
Foward Primer	5
Reverse Primer	5
DNA polymerase TAQ	1
Thermopol Buffer	5
dH₂O	28
TOTAL	50μL

95°C,5min -> ( 95°C,1min -> 52°C,1min -> 72°C,1min )･･･25cycles -> 72°C,10min -> 6°C

--->Gel Check

Sample DNA	3
Loading Dye	2
dH₂O	7
TOTAL	12μL

From left;

BBa_C0078(2007) -> None
BBa_C0078(2006) -> None
BBa_C0076(2007) -> None
BBa_C0070(2007) -> OK
BBa_C0070(2006) -> OK

Transformation

competent cells : XL10G

BBa_S03154(2007)
BBa_S03154(2006)
BBa_I9026(2007)
BBa_I9026(2006)
BBa_I9030(2007)
BBa_I9030(2006)

--->(23/8)Digestion

Team:Output

Transformation

BBa_J32007(2007)-->no colony-->no colony(2 times)-->colony(3times)
BBa_B0034(2007)-->colony

@@ Line 25: / Line 25: @@
 #Inoculated (Reporter) culture from glycerol stock(-80&deg;C freezer) in 2mL LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL mediumfor 12h, at 37&degC.
-#We diluted 10<sup>5</sup>-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other. we cultured for 12h at 37&deg;C.
+#We diluted 10<sup>5</sup>-fold, and plated 20ul of the resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.
-<br>37°Cで12h培養後、きちんとコロニーができていることを確認し、両方のプレートにUVを照射する。wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
+#We cultured for 12h at 37&deg;C.
-<br>UVを照射して9h後、21h後にそれぞれ両方のプレートに次の同じ操作をする。
+#We iraddiated UV each plate. Wavelength:254nm,distance from the UV lamp to the cultures were 14cm.
-<br>両方のプレートのコロニーをつついてそれぞれ(LB-Amp,Cm),(LB-Amp,Cm,AHL100nM)2ml培養する。
+#UVを照射して9h後、21h後にそれぞれ両方のプレートに次の同じ操作をする。
-<br>37°C,で12h培養後、濁っていたので、それぞれを(LB-Amp,Cm),(LB-Amp,Cm,AHL100nM)のプレートに10<sup>5</sup>希釈して20μl撒いた。
+#両方のプレートのコロニーをつついてそれぞれ(LB-Amp,Cm),(LB-Amp,Cm,AHL100nM)2ml培養(37&deg;C,12h)する。
+#We diluted 10<sup>5</sup>-fold, and plated 20ul of the
+resulting solution to (LB-Amp,Cm)(LB-Amp,cm,AHL100nM) plates each other.