Team:Chiba/Calendar-Home/30 August 2008

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29 August 2008 <|> 31 August 2008

Contents

Laboratory work

Team:Input

Digestion

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

混ぜ表

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

-->30μlゲル抽出

-->ゲル抽出

-->Zymo Clean


5μ抽出,うち1μlをゲルチェック-->OK,4μlはLigation用


Digestion

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

混ぜ表

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

-->30μlゲル抽出

ゲル抽出

-->Zymo Clean

-->SAP


混ぜ表

dH2O 9
DNA 10
SAP 1
SAP Buffer 10
TOTAL 30

5μ抽出,うち1μlをゲルチェック

-->OK,4μlはLigation用


Ligation

混ぜ表

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

-->Transformation(XL10G)

-->CFU40(b.gは22)


コロニーPCR(16コつつく):PCR


混ぜ表

VF 2
VFR 2
dNTP 2
thermo pol buffer 2
Tag 0.3
dH2O 11.7
TOTAL 20

-->Insertチェックしたら1つだけ正しい位置にバンドが出現。そのコロニーを2ml培養し、mini prepした。

-->Mini prep



Digestion

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

混ぜ表

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

Gel Extract

混ぜ表

Digestion産物 30
Dye 6
TOTAL 36

-->Zymo Clean

-->NFW5μlで溶出

-->Gel Check


混ぜ表

dH2O 4
DNA 1
Dye 1
TOTAL 6

-->Gel Check-->OK


Digestion

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

混ぜ表

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

Gel Extract

混ぜ表

Digestion産物 30
Dye 6
TOTAL 36

-->Zymo Clean

-->NFW10μlで溶出


-->SAP


混ぜ表

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

-->37℃で1h、乾燥機(65℃)で15min,

-->Binding Bufferを90μl加え、VORTEX -->Zymo Clean

-->NFW5μlで抽出

Gel Check


混ぜ表

dH2O 4
DNA 1
Dye 1
TOTAL 6

-->ゲルチェックOK

Ligation

混ぜ表

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

-->25℃で2h放置 -->Transformation(XL10G):

-->CFU40(b.gは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:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500


--->Digestion Test
  • insert:C0170 + vector:J04500 -> Sample No.1~4
  • insert:C0178 + vector: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
TOTAL1010


--->Gel Check
Chiba-0830.JPG
Sample No. 1~910~1819~27
Sample DNA 11010
Loading Dye 122
dH2O 4--
TOTAL 61212
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](rLuc)
Sample No. [http://partsregistry.org/Part:BBa_J52008 BBa_J52008]
DNA tamplate 1
rLuc_fwd 2.5
Rev primer 2.5
Thermo pol Buffer 5
dNTPmix 5
Vent pol 0.5
dH2O 34
TOTAL 50
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