Team:Chiba/Calendar-Home/30 August 2008

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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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