Team:Chiba/Calendar-Home/21 August 2008

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Revision as of 01:08, 30 October 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.

[http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2007)
[http://partsregistry.org/Part:BBa_R0051 BBa_R0051](2006)
[http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2007)
[http://partsregistry.org/Part:BBa_J06650 BBa_J06650](2006)
[http://partsregistry.org/Part:BBa_J22136 BBa_J22136](2007)
[http://partsregistry.org/Part:BBa_J22141 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 ---
- (Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-
- (Reporter)-PcI-GFP-p15a-Cm-(BW)

Inoculated (Reporter) culture from glycerol stock(-80°C freezer) in 2mL LB medium.(LB-Amp,Cm)(LB-Amp,Cm,AHL100nM)

Inoculated transformants for 12 hours in 2ml LB-Ampicillin, Chloramphenicol medium and LB-Ampicillin, Chloramphenicol, 100nmAHL medium .(37&ged;C, 12h).　それぞれを10⁵希釈してAHLが入っていないものはLB-Amp,Cm,AHLが入っているものはAHL-Amp,Cm,AHL100nMのプレートにそれぞれ20μl撒く。
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。UVの波長は254nm,UVランプとプレートの距離は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培養する。
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10⁵希釈して20μl撒いた。

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

[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007)
[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006)
[http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007)
[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007)
[http://partsregistry.org/Part:BBa_C0078 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;

[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2007) -> None
[http://partsregistry.org/Part:BBa_C0078 BBa_C0078](2006) -> None
[http://partsregistry.org/Part:BBa_C0076 BBa_C0076](2007) -> None
[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2007) -> OK
[http://partsregistry.org/Part:BBa_C0070 BBa_C0070](2006) -> OK

Transformation

competent cells : XL10G

[http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
[http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2006)
[http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
[http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2006)
[http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2007)
[http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)

--->(23/8)Digestion

Team:Output

Transformation

[http://partsregistry.org/Part:BBa_J32007 BBa_J32007](2007)-->no colony-->no colony(2 times)-->colony(3times)
[http://partsregistry.org/Part:BBa_B0034 BBa_B0034](2007)-->colony

@@ Line 19: / Line 19: @@
-*UV irradiation test  (plate version)
+*UV irradiation test  (plate phase)
 *two plasmids from ---
 **(Repressor)-Ptrc-LuxR-Plux-cI-colE1-Amp-