Team:Chiba/jk/β/week 2

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>送受信班

>Protocols


Contents

Week 2

>last week

24 August, 2008

(22/8)--->Mini prep
  1. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
  2. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)
  3. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)
  4. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006)


--->Digestion test
  1. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
  2. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006)
  3. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007)
  4. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006)


Sample No. 5678
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) 1---
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -1--
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) --1-
[http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) ---1
EcoRⅠ 0.10.10.10.1
Buffer 4 0.90.90.90.9
dH2O 8888
TOTAL 10101010


--->Gel Check
Chiba-0824-1.JPG
Sample No. 1~8
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  1. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> ?
  2. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
  3. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> OK
  4. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> OK
  5. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
  6. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
  7. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> OK
  8. [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> None?
Chiba-0824-2.JPG
Sample No. 5~8
Sample DNA 9
Loading Dye 2
dH2O 1
TOTAL 12

From the left; （single digestuion）

5, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
6, [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2006) -> OK
7, [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2007) -> 3400bp:Bad, 2200bp:OK
8, [http://partsregistry.org/Part:BBa_J13002 BBa_J13002](2006) -> OK

25 August, 2008

(24/8)--->Digestion
  1. Double Digestion:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
  2. Double Digesiton:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007)
  3. Single Digestion:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007)
  4. [http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007)
Sample No. 1234
Sample DNA 5511
EcoRⅠ 0.50.5--
SpeⅠ 0.50.5--
Buffer 1 11--
Buffer 3 --1-
BSA 11--
dH2O 227.5-
TOTAL 1010101
--->Gel Check
Chiba-0825-1.JPG
Sample No. 1~34
Sample DNA 101
Loading Dye 21
dH2O -4
TOTAL 126
From right;
  1. Double Digestion:[http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007) -> OK
  2. Double Digesiton:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007) -> ?(low)
  3. Single Digestion:[http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007) -> ?(low)
  4. [http://partsregistry.org/Part:BBa_R0010 BBa_R0010](2007) -> OK(low)


--->Digestion (Double Digestion)

  1. [http://partsregistry.org/Part:BBa_C0178 BBa_C0178]
  2. [http://partsregistry.org/Part:BBa_C0170 BBa_C0170]
  3. [http://partsregistry.org/Part:BBa_J04500 BBa_J04500](2007)
Sample No. 123
Sample DNA 33ng/μl×60μl33ng/μl×60μl100ng/μl×20μl
PstⅠ 222
XbaⅠ 22-
SpeⅠ --2
Buffer 2 --5
Buffer 3 1010-
BSA 10105
dH2O 161616
TOTAL 10010052

26 August, 2008

(8/25)--->Gel Check
Chiba-0825-2.JPG
Sample No. 1~3
Sample DNA 30
Loading Dye 6
TOTAL 36
1, BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
Insert:609bp -> OK
Chiba-0825-3.JPG
2, BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
Insert:609bp -> OK
Chiba-0825-4.JPG
3, BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
-> OK


--->Gel Extract


--->Zymo Clean
  1. 5μl
  2. 5μl
  3. 27μl


--->Gel Check
Chiba-0825-5.JPG
Sample No. 1~3
Sample DNA 2
Loading Dye 2
H2O 8
TOTAL 12
From left;
  1. insert:C0178 -> OK
  2. insert:C0170 -> OK?
  3. vector:J04500 -> OK


--->SAP
Sample No. 3
Sample DNA 26
SAP 1
SAP Buffer 3
TOTAL 30μL


--->Zymo Clean
Sample No.3->9μl


--->Gel Check
Chiba-0826.JPG
Sample No.3 1
Loading Dye 1
dH2O 4
TOTAL 6
Sample No.3 -> OK ･･･30ng?


--->Ligation
]

</tr>

Sample No. (1)(2)(3)(4)(5)
insert①C0178 1--1-
insert②C0170 -1--1
vector③J04500 --222
ligase 11111
Buffer 22222
dH2O 66544
TOTAL 1010101010


--->Transformation
  1. insert①C0178
  2. insert②C0170
  3. vector③J04500
  4. insert①C0178+vector③J04500
  5. insert②C0170+vector③J04500


27 August, 2008

Digestion Test

Sample DNA : BBa_R0010[http://partsregistry.org/Part:BBa_R0010]
Sample No. 123
Sample DNA 111
PstⅠ --0.5
EcoRⅠ -0.50.5
Buffer 3 -11
BSA --1
dH2O -7.52
TOTAL 11010


--->Gel Check
Chiba-0827.JPG
Sample No. 123
Sample DNA 155
Loading Dye 111
H2O 4--
TOTAL 666
From left;
  1. BBa_R0010
  2. Single Digestion
  3. Double Digestion


(26/8)--->Colony-PCR
Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 5
Reverse Primer 5
DNA polymerase 0.5
Thermopol Buffer 5
dH2O 28.5
TOTAL 50μL


95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃



--->Gel Check
Chiba-0827-2.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
1~5 insert:C0170 + vector:J04500 -> OK
6 F2620･･Positive Control -> OK
7~11 insert:C0178 + vector:J04500 -> OK


--->(28/8)Miniprep


Transformation

competent cells : XL10G
  • BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
  • BBa_C0062(2006) [http://partsregistry.org/Part:BBa_C0062] -> no colony on plates
  • BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
  • BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
  • BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
  • BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
  • BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
  • BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
  • BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
  • BBa_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]


--->Mini prep


--->(29/8)Gel Check


28 August, 2008

(27/8)--->Mini prep
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500


--->Digestion Test
  1. insert:C0170 + vector:J04500
  2. insert:C0178 + vector:J04500
Sample No. 3456
insert:C0170 + vector:J04500 1-1-
insert:C0178 + vector:J04500 -1-1
EcoRⅠ 0.10.10.10.1
SpeⅠ --0.10.1
Buffer 3 1111
BSA --11
dH2O 7.87.86.86.8
TOTAL 10101010


--->(29/8)Gel Check
Chiba-0828.JPG
Sample No. 1,23~6
Sample DNA 110
Loading Dye 12
H2O 4-
TOTAL 612
From left;
  1. 　-> OK
  2. 　-> OK
  3. 　-> OK
  4. 　-> OK
  5. 　-> OK
  6. 　-> OK

29 August, 2008

--->Digestion Test
  1. BBa_R0010(2007) [http://partsregistry.org/Part:BBa_R0010]
  2. BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
  3. BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
  4. BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
  5. BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
  6. BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
  7. BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
  8. BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
  9. BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
  10. BBa_S03160(2006) [http://partsregistry.org/Part:BBa_S03160]


Sample No. S1~10W1W2~10
Sample DNA 142
PstⅠ -0.0750.1
EcoRⅠ 0.10.0750.1
Buffer 3 10.751
BSA -0.751
dH2O 7.94.355.8
TOTAL 101010


--->Gel Check
Chiba-0829.JPG
Sample No. 1~10S1~S10,W1~W10
Sample DNA 110
Loading Dye 12
TOTAL 612
From left;
1~5,S1,S5,W1~W5
Chiba-0829-2.JPG
From left;
6~10,S6~S10,W6~W10


(27/8)--->Gel Check
Chiba-0829-3.JPG
Sample DNA 0.5
Loading Dye 1
dH2O 4.5
TOTAL 6μL
R0010 -> OK


Digestion

  1. BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  2. BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
  3. BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  4. BBa_R0010(2006) [http://partsregistry.org/Part:BBa_R0010]


Sample No. 1~34
Sample DNA 100ng/μL×20200ng/μL×30
PstⅠ 22
XbaⅠ 2-
SpeⅠ -2
Buffer 2 -5
Buffer 3 5-
BSA 55
dH2O 166
TOTAL 5050
--->Gel Check
Chiba-0829-4.JPG
Sample No. 1~34
Sample DNA 103
Loading Dye 22
dH2O -7
TOTAL 1212
insert-I9026 : 685bp -> None
Chiba-0829-5.JPG
insert-I9030 : 745bp -> None
Chiba-0829-6.JPG
insert-S03154 : 685bp -> None
Chiba-0829-7.JPG
vector-R0010 : 2079bp -> OK


--->Gel Extract


--->Zymo Clean
->15μL
--->SAP
Sample No. 4
Sample DNA 15
SAP 2
SAP Buffer 3
dH2O 10
TOTAL 30μL


--->Zymo Clean
--->Suspension! (We got information, by the team-output, to the effect that the density of the sample is too low.)


30 August, 2008

Transformation
competent cells : XL10G
  • BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
  • BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
  • BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
  • BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
  • BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
  • BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
  • BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
  • BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
--->(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
  • BBa_T9002(2007)[http://partsregistry.org/Part:BBa_T9002] -> 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??

>next week

ホーム メンバー紹介 プロジェクト紹介 Parts Submitted to the Registry モデリング ノート
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