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

From 2010.igem.org

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

Latest revision as of 18:08, 27 October 2010


E.coli Fiber Project Notebook

August 2010
SUNMONTUEWEDTHUFRISAT
1 2 3 4 5 6 7
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22 23 24 25 26 27 28
29 30 31
September 2010
SUNMONTUEWEDTHUFRISAT
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
October 2010
SUNMONTUEWEDTHUFRISAT
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

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
  1. mix materials(DNA<50ng)
  2. PCR
  3. Add EDTA and Ethanol and put at room temperature　(15min)
  4. centrifuge (8000rpm,30min)
  5. throw away supernatant
  6. add ethanol again
  7. centrifuge (8000rpm,15min)
  8. put at refrigerator
  9. throw away supernatant
  10. add Hi-Di solution
  11. transfer these sample to plate for sequence
  12. read sequence



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