Team:Chiba/Calendar-Home/30 August 2008

From 2008.igem.org

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x-RBS-cI-s-p(780bp)
x-RBS-cI-s-p(780bp)
-
混ぜ表
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
Line 65: Line 64:
-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)
-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
Line 103: Line 102:
-->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
-->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
-
eluted with ?L of NFW.
+
eluted with 10mL of NFW.
-->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
-->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="57"></td>
<td width="57"></td>
Line 144: Line 143:
'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
-
混ぜ表
+
 
<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
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-->'''[[Team:Chiba/protocol/transformation|Transformation]]'''(XL10G)
-->'''[[Team:Chiba/protocol/transformation|Transformation]]'''(XL10G)
-
-->CFU40(b.gは22)
+
-->CFU40(b.g:22)
'''[[Team:Chiba/protocol/PCR|PCR]]''' of 16 colonies)
'''[[Team:Chiba/protocol/PCR|PCR]]''' of 16 colonies)
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
Line 216: Line 215:
</table>
</table>
-
-->[[Team:Chiba/protocol/gelcheck|Gel Check]]?
+
-->[[Team:Chiba/protocol/gelcheck|Gel Check]]
-
result:One colony was apparent.(1つだけ正しい位置にバンドが出現。)
+
result:One colony was apparent.
Picked the colony and incubate in 2mL of LB.
Picked the colony and incubate in 2mL of LB.
Line 230: Line 229:
x-RBS-cI-s-p(780bp)
x-RBS-cI-s-p(780bp)
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
Line 266: Line 265:
'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="57"></td>
<td width="57"></td>
Line 291: Line 290:
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="57"></td>
<td width="57"></td>
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-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)
-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
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'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="57"></td>
<td width="57"></td>
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-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="57"></td>
<td width="57"></td>
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</tr>
</tr>
</table>
</table>
-
-->left for an hour at 37 degrees,for 15 minutes at 65 degrees.（乾燥機・・・）
+
-->left for an hour at 37 degrees,for 15 minutes at 65 degrees.
-->added 90μL Binding Buffer and vortexed.
-->added 90μL Binding Buffer and vortexed.
Line 418: Line 417:
-
混ぜ表
+
 
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="150" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="57"></td>
<td width="57"></td>
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'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
-
混ぜ表
+
 
<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="200" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<td width="257"></td>
<td width="257"></td>
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</tr>
</tr>
</table>
</table>
-
-->left at rest at room temparature(25　degrees?本当に?)
+
-->left at rest at room temparature(25　degrees)
-->'''[[Team:Chiba/protocol/transformation|Transformation]]'''(XL10G):
-->'''[[Team:Chiba/protocol/transformation|Transformation]]'''(XL10G):
-
-->CFU40(b.gは22)
+
-->CFU40(b.g:22)
===Team:Communication===
===Team:Communication===
Line 494: Line 493:
:(29/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
:(29/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
-
::#insert:C0170 + vector:J04500
+
::#insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
-
::#insert:C0178 + vector:J04500
+
::#insert:[http://partsregistry.org/Part:BBa_C0178 BBa_C0178] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
-
::*insert:C0170 + vector:J04500 -> Sample No.1~4
+
::#insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500] -> Sample No.1~4  
-
::*insert:C0178 + vector:J04500 -> Sample No.5~8
+
::#insert:[http://partsregistry.org/Part:BBa_C0178 BBa_C0178] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500] -> Sample No.5~8
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](2007) -> Sample No.9
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](2007) -> Sample No.9
::*Single Digestion of Sample No.1~9 -> Sample No.10~18
::*Single Digestion of Sample No.1~9 -> Sample No.10~18
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</tr>
</tr>
<tr>
<tr>
-
<td>XbaⅠ</td><td>0.1</td><td>0.1</td>
+
<td>XbaI</td><td>0.1</td><td>0.1</td>
</tr>
</tr>
<tr>
<tr>
-
<td>SpeⅠ</td><td>-</td><td>0.1</td>
+
<td>SpeI</td><td>-</td><td>0.1</td>
</tr>
</tr>
<tr>
<tr>
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</tr>
</tr>
<tr>
<tr>
-
<td>TOTAL</td><td>10</td><td>10</td>
+
<td>TOTAL</td><td>10μl</td><td>10μl</td>
</tr>
</tr>
</table>
</table>
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<tr>
<tr>
<td>TOTAL</td>
<td>TOTAL</td>
-
<td>6</td><td>12</td><td>12</td>
+
<td>6μl</td><td>12μl</td><td>12μl</td>
</tr>
</tr>
</table>
</table>
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::23 -> OK??
::23 -> OK??
|}
|}
-
 
===Team:Output===
===Team:Output===
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[[Team:Chiba/protocol/PCR|PCR]]
[[Team:Chiba/protocol/PCR|PCR]]
-
*[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]①
+
*[http://partsregistry.org/Part:BBa_J52008 BBa_J52008](1)
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<table width="250" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
<tr>
<tr>
<td width="257">Sample No.</td>
<td width="257">Sample No.</td>
-
<td>①</td>
+
<td>1</td>
</tr>
</tr>
<tr>
<tr>

Latest revision as of 23:13, 29 October 2008

>Home | Notebook

29 August 2008 <|> 31 August 2008


Contents

Meeting with UmeG

the ppt (in Japanese) for our weekly meeting

  1. team-input
  2. team-communication
  3. team-output

Laboratory work

Team:Input

Digestion

x-RBS-cI-s-p(780bp)

double
dH2O(μL) 6
10×BSA(μL) 10
10×NE(μL) 10
PstI(μL) 2
XbaI(μL) 2
DNA(μL) 33.6ng/μl×30μl(1μg)
TOTAL(μL) 60

-->30μlGel extraction

-->Zymo Clean

eluted with 5μL of Nuclease free water(NFW)

Gel Check(1μl)

-->OK

(remains of the solution(4μL)-->Ligation)

Digestion

-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)


double
dH2O(μL) 4
10×BSA(μL) 10
10×NE(μL) 10
PstI(μL) 2
SpeI(μL) 4
DNA(μL) 75ng/μl×30μl(2μg)
TOTAL(μL) 60

-->30μlGel extraction

-->Zymo Clean

eluted with 10mL of NFW.

-->SAP


dH2O(μL) 9
DNA(μL) 10
SAP(μL) 1
SAP Buffer(μL) 10
TOTAL(μL) 30

eluted with 5μL of Nuclease free water(NFW)

Gel Check(1μl)

-->OK

(remains of the solution(4μL)-->Ligation)


Ligation


-b.g
dH2O(μL) 1.48
Ligase(μL) 11
Ligase Buffer(μL) 22
Vector(μL) 1.81
Insert(μL) 3-
TOTAL(μL) 1010

-->Transformation(XL10G)

-->CFU40(b.g:22)


PCR of 16 colonies)


VF(μL) 2
VR2(μL) 2
dNTP(μL) 2
thermo pol buffer(μL) 2
Tag(μL) 0.3
dH2O(μL) 11.7
TOTAL(μL) 20

-->Gel Check

result:One colony was apparent.

Picked the colony and incubate in 2mL of LB.

-->Mini prepped


Digestion

x-RBS-cI-s-p(780bp)


double
dH2O(μL) 6
10×BSA(μL) 10
10×NE(μL) 10
PstI(μL) 2
XbaI(μL) 2
DNA(μL) 33.6ng/μl×30μl(1μg)
TOTAL(μL) 60

Gel Extract


Digested DNA Sample(μL) 30
Loading Dye(μL) 6
TOTAL(μL) 36

-->Zymo Clean

-->eluted with 5μl of NFW

-->Gel Check


dH2O(μL) 4
DNA(μL) 1
Loading Dye(μL) 1
TOTAL(μL) 6

-->Gel Check-->OK


Digestion

-e-x-Ptet-s-p-(PMB1,Amp)(Insert:54bp,Vector:2079bp)


double
dH2O(μL) 4
10×BSA(μL) 10
10×NE(μL) 10
PstI(μL) 2
SpeI(μL) 4
DNA(μL) 75ng/μl×30μl(2.25μg)
TOTAL(μL) 60

Gel Extract


Digested DNA Sample(μL) 30
Loading Dye(μL) 6
TOTAL(μL) 36

-->Zymo Clean

-->eluted with 10μl of NFW


-->SAP


dH2O(μL) 9
DNA(μL) 10
SAP(μL) 1
SAP Buffer(×10)(μL) 10
TOTAL(μL) 30

-->left for an hour at 37 degrees,for 15 minutes at 65 degrees.

-->added 90μL Binding Buffer and vortexed.

-->Zymo Clean

-->eluted with 5μl of NFW

Gel Check


dH2O(μL) 4
DNA(μL) 1
Loading Dye(μL) 1
TOTAL(μL) 6

-->Gel Check-->OK

Ligation


-b.g
dH2O(μL) 1.48
Ligase(μL) 11
Ligase Buffer(μL) 22
Vector(μL) 1.81
Insert(μL) 3-
TOTAL(μL) 1010

-->left at rest at room temparature(25　degrees) -->Transformation(XL10G):

-->CFU40(b.g:22)

Team:Communication

Transformation
competent cells : XL10G
  • [http://partsregistry.org/Part:BBa_I9026 BBa_I9026](2007)
  • [http://partsregistry.org/Part:BBa_I9030 BBa_I9030](2006)
  • [http://partsregistry.org/Part:BBa_S03154 BBa_S03154](2007)
  • [http://partsregistry.org/Part:BBa_S03156 BBa_S03156](2007)
  • [http://partsregistry.org/Part:BBa_S03158 BBa_S03158](2007)
  • [http://partsregistry.org/Part:BBa_S03160 BBa_S03160](2007)
  • [http://partsregistry.org/Part:BBa_C0062 BBa_C0062](2007)
  • [http://partsregistry.org/Part:BBa_C0179 BBa_C0179](2007)
--->(31/8)Mini prep


(29/8)--->Mini prep
  1. insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]
  2. insert:[http://partsregistry.org/Part:BBa_C0178 BBa_C0178] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]


--->Digestion Test
  1. insert:[http://partsregistry.org/Part:BBa_C0170 BBa_C0170] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500] -> Sample No.1~4
  2. insert:[http://partsregistry.org/Part:BBa_C0178 BBa_C0178] + vector:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500] -> Sample No.5~8
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002](2007) -> Sample No.9
  • Single Digestion of Sample No.1~9 -> Sample No.10~18
  • Double Digestion of Sample No.1~9 -> Sample No.19~27
Sample No.Single : 10~18Double : 19~27
Sample DNA15
XbaI0.10.1
SpeI-0.1
Buffer 211
BSA11
dH2O6.92.8
TOTAL10μl10μl


--->Gel Check
Chiba-0830.JPG
Sample No. 1~910~1819~27
Sample DNA 11010
Loading Dye 122
dH2O 4--
TOTAL 6μl12μl12μl
From left;
Sanple No.1~13
-> OK
Chiba-0830-2.JPG
From left;
Sample No.14~17,24~16
14 -> OK
15,16 -> Bad
17 -> None
24~16 -> Bad
Chiba-0830-3.JPG
From left;
Sample No.18,27,19~23
18,27 -> Bad
19~22 -> OK
23 -> OK??

Team:Output

Transformation

  • mcherry

PCR

  • [http://partsregistry.org/Part:BBa_J52008 BBa_J52008](1)
Sample No. 1
DNA tamplate 1
rLuc_fwd 2.5
Rev primer 2.5
Thermo pol Buffer 5
dNTPmix 5
Vent pol 0.5
dH2O 34
TOTAL 50μl
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