Team:Tokyo Metropolitan/Notebook/Fiber/2010/10/24

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Latest revision as of 18:08, 27 October 2010

E.coli Fiber Project Notebook

2010/10/24 Sunday (Naoto)

member

naoto and watachin

Preparation:making preculture for Miniprep

material

colony of E.coli
- bcsA→No.21,22
- bcsB→No.10
- bcsC→No.54,64
- bcsD→No.75
LB+Cam broth

procedure

see protocol2

Experiment:Ligation

material

see protocol8

vector
- pUC19(digested by XbaI)
Insert
- bcsA(digested by XbaI+SpeI)
- bcsB(digested by XbaI+SpeI)
- bcsC(digested by XbaI+SpeI)
- bcsD(digested by XbaI+SpeI)

procedure

refer to　protocol8

Experiment:Transformation

material

see protocol9

Ecos JM109/Nova Blue(Competent Cell)

procedure

refer to　protocol9

Experiment:Miniprep

material

see protocol10

Preculture

No.21,22:bcsA

No.10:bcsB

No.54,64:bcsC

No.75:bcsD

procedure

refer to protocol10

Experiment:Sequence

material

PCR
- DNA(No.21,22,10,54,64,75:following before "Miniprep")
- Big Dye
- primer
- DW
Ethanol precipitation
- ethanol
- EDTA

procedure

mix materials(DNA<50ng)
PCR
Add EDTA and Ethanol and put at room temperature　(15min)
centrifuge (8000rpm,30min)
throw away supernatant
add ethanol again
centrifuge (8000rpm,15min)
put at refrigerator
throw away supernatant
add Hi-Di solution
transfer these sample to plate for sequence
read sequence

@@ Line 1: / Line 1: @@
 {{:Team:Tokyo_Metropolitan/Notebook/Fiber}}
+__NOTOC__
 ==2010/10/24 Sunday (Naoto)==
-'''member'''
+:'''member'''
-naoto and watachin
+:naoto and watachin
-===Experiment:Colony PCR===
+===Preparation:making preculture for Miniprep ===
-'''material'''
+:'''material'''
-*colony of E.coli
+:*colony of ''E.coli''
-**bcsB:1~32
+:**bcsA→No.21,22
-**bcsC:33~64
+:**bcsB→No.10
-**bcsD:65~96
+:**bcsC→No.54,64
-*other materials were same as <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80PCR">protocol3</a></html>
+:**bcsD→No.75
+:*LB+Cam broth
-'''result'''
+:'''procedure'''
-From the length of bands
+:see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80Grow up culture of E.coli">protocol2</a></html>
-bcsC→No.54 and 64
-bcsD→No.75
-seem to be correct inserts
 ===Experiment:Ligation===
-'''material'''
+:'''material'''
-see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80ligation">protocol8</a></html>
+:see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80ligation">protocol8</a></html>
-*vector
+:*vector
-**pUC19(digested by XbaI)
+:**pUC19(digested by XbaI)
-*Insert
+:*Insert
-**bcsA(digested by XbaI+SpeI)
+:**bcsA(digested by XbaI+SpeI)
-**bcsB(digested by XbaI+SpeI)
+:**bcsB(digested by XbaI+SpeI)
-**bcsC(digested by XbaI+SpeI)
+:**bcsC(digested by XbaI+SpeI)
-**bcsD(digested by XbaI+SpeI)
+:**bcsD(digested by XbaI+SpeI)
-'''procedure'''
+:'''procedure'''
-refer to　<html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80ligation">protocol8</a></html>
+:refer to　<html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80ligation">protocol8</a></html>
 ===Experiment:Transformation===
-'''material'''
+:'''material'''
-see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80transformation">protocol9</a></html>
+:see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80transformation">protocol9</a></html>
-*Competent cell
+:*Ecos JM109/Nova Blue(Competent Cell)
-**Ecos JM109/Nova Blue
-'''procedure'''
+:'''procedure'''
-refer to　<html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80transformation">protocol9</a></html>
+:refer to　<html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80transformation">protocol9</a></html>
 ===Experiment:Miniprep===
-'''material'''
+:'''material'''
-see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80miniprep">protocol10</a></html>
+:see <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80miniprep">protocol10</a></html>
-*Preculture
+:*Preculture
-No.21,22:bcsA
+:No.21,22:bcsA
-No.10:bcsB
+:No.10:bcsB
-No.54,64:bcsC
+:No.54,64:bcsC
-No.75:bcsD
+:No.75:bcsD
-'''procedure'''
+:'''procedure'''
-refer to <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80miniprep">protocol10</a></html>
+:refer to <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80miniprep">protocol10</a></html>
 ===Experiment:Sequence===
-'''material'''
+:'''material'''
-*PCR
+:*PCR
-**DNA(No.21,22,10,54,64,75:following before "Miniprep")
+:**DNA(No.21,22,10,54,64,75:following before "Miniprep")
-**Big Dye
+:**Big Dye
-**primer
+:**primer
-**DW
+:**DW
-*Ethanol precipitation
+:*Ethanol precipitation
-**ethanol
+:**ethanol
-**EDTA
+:**EDTA
+:'''procedure'''
+:#mix materials(DNA<50ng)
+:#PCR
+:#Add EDTA and Ethanol and put at room temperature　(15min)
+:#centrifuge (8000rpm,30min)
+:#throw away supernatant
+:#add ethanol again
+:#centrifuge (8000rpm,15min)
+:#put at refrigerator
+:#throw away supernatant
+:#add Hi-Di solution
+:#transfer these sample to plate for sequence
+:#read sequence
-'''procedure'''
+<br/>
-#mix materials(DNA<50ng)
-#PCR
-#Add EDTA and Ethanol and put at room temperature　(15min)
-#centrifuge (8000rpm,30min)
-#throw away supernatant
-#add ethanol again
-#put at refrigerator
-#throw away supernatant