Team:Chiba/jk/β/week 3

From 2008.igem.org

(Difference between revisions)
(→>next week)
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<tr>
<tr>
<td width="257">Sample</td>
<td width="257">Sample</td>
-
<td>Single Digesiton 2~5</td><td>Double Digestion 2~5</td><td>Single Digestion T9002①</td><td>Single Digesiton T9002②</td><td><Double Digestion T9002①②</td>
+
<td>Single Digesiton 2~5</td><td>Double Digestion 2~5</td><td>Single Digestion T9002①</td><td>Single Digesiton T9002②</td><td>Double Digestion T9002①②</td>
</tr>
</tr>
<tr>
<tr>
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::*BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
::*BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
|}
|}
 +
===1 September, 2008===
===1 September, 2008===
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|}
|}
-
:--->zymo
+
:--->'''[[Team:Chiba/protocol/ligation/gelex|Gel extract]]'''
 +
 
 +
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|zymo]]'''
 +
::insert-1(I9026) -> 7μL
 +
::insert-2(I9030) -> 7μL
 +
::insert-3(S03154) -> 7μL
 +
::vector-4(R0010) -> 15μL
 +
 
 +
:--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|SAP]]'''
::vector-4(R0010)
::vector-4(R0010)
-
::-> 15μL
 
-
:--->SAP
+
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo]]'''
-
::vectr-4(R0010)
+
::vector-4(R0010) -> 20μL
-
:--->Zymo
 
-
::1 -> 7μL
 
-
::2 -> 7
 
-
::3 -> 7
 
-
::4 -> 20
 
 +
:--->
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
:Competent cells : BW ΔFliC  40μL
:Competent cells : BW ΔFliC  40μL
-
::*Plac+RBS+LasI : Sample No.L1~L4
+
::*BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007] : Sample No.L1~L4
-
::*Plac+RBS+RhlI : Sample No.R1~R4
+
::*BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008] : Sample No.R1~R4
:--->(2/9)OD
:--->(2/9)OD

Revision as of 07:49, 26 September 2008

>送受信班

Contents

Week 3

>last week

31 August, 2008

Transformation
Competent Cells : XL10G
  • Plac+RBS+LasI
  • Plac+RBS+RhlI
---> We saved these plates with a refrigerator.


Digestion

  1. BBa_I9026[http://partsregistry.org/Part:BBa_I9026](2007)
  2. BBa_I9030[http://partsregistry.org/Part:BBa_I9030](2006)
  3. BBa_S03154[http://partsregistry.org/Part:BBa_S03154](2007)
  4. BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
Sample No 1~34
Sample DNA1210
PstⅠ 0.20.2
XbaⅠ0.2-
SpeⅠ-0.4
Buffer 2 -1.5
Buffer 3 2-
BSA 21.5
dH2O 3.61.4
TOTAL 2015


--->(1/9)Gel Check


(30/8)--->Mini prep


--->Digestion test
  • Plac+RBS+RhlI Sample No.2~5
  • BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
Sample Single Digesiton 2~5Double Digestion 2~5Single Digestion T9002①Single Digesiton T9002②Double Digestion T9002①②
Sample DNA13313
XbaⅠ0.10.10.10.10.1
SpeⅠ-0.1--0.1
Buffer 2 0.90.80.90.90.8
BSA 11111
dH2O 75575
TOTAL 1010101010


--->Gel Check

Chiba-0831-p87.JPG
Sample No Plac+RBS+RhlI Sample No.2~5, BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①② Single & Double Digestion
Sample DNA 110
Loading Dye 12
dH2O 4-
TOTAL 612
From Left
  • Single Digestion Plac+RBS+RhlI 2~5
  • Single Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
  • Double Digestion Plac+RBS+RhlI 2~5
  • Double Digestion BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②
  • BBa_T9002[http://partsregistry.org/Part:BBa_T9002]①、②


1 September, 2008

(31/8)--->Gel Check
Chiba-0901.JPG
Sample No 1~34
Sample DNA 2015
Loading Dye 43
TOTAL 2418
From left;
insert-1(I9026)
insert-2(I9030)
Chiba-0901-2.JPG
insert-3(S03154)
Chiba-0901-3.JPG
vector-4(R0010)
--->Gel extract
--->zymo
insert-1(I9026) -> 7μL
insert-2(I9030) -> 7μL
insert-3(S03154) -> 7μL
vector-4(R0010) -> 15μL
--->SAP
vector-4(R0010)
--->Zymo
vector-4(R0010) -> 20μL


--->


Transformation

Competent cells : BW ΔFliC 40μL
  • BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007] : Sample No.L1~L4
  • BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008] : Sample No.R1~R4
--->(2/9)OD

2 September, 2008

(31/8)--->Gel Check
Chiba-0902.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
I9026
I9030
S03154


(1/9)--->Colony-PCR
Colony PCR of 8 colonies from ligation plates (1/9-(1),(2)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 0.3
Reverse Primer 0.3
DNA polymerase TAQ 0.5
Thermopol Buffer 3
dH2O 20.5
TOTAL 30μL


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


--->Gel Check

Chiba-0902-2.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • Plac+RBS+RhlI+LVA
R1 -> OK
R2 -> Bad
R3~R7 -> OK
R8 -> Bad
Chiba-0902-3.JPG
From left;
  • Plac+RBS+CinI+LVA
C1,C2 -> OK
C3 -> Bad
C4~C6 -> OK
Chiba-0902-4.JPG
From left;
  • Plac+RBS+CinI+LVA
C7,C8 -> OK
  • BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->OD測


Transformation

Competent cells : XL10G 30μL
  • C0161(2007) [http://partsregistry.org/Part:BBa_C0161]
  • C0161(2006) [http://partsregistry.org/Part:BBa_C0161]
  • C0261(2007) [http://partsregistry.org/Part:BBa_C0261]
  • C0261(2006) [http://partsregistry.org/Part:BBa_C0261]
--->(4/9)Mini prep

3 September, 2008

(2/9)--->Mini prep
--->Gel Check
Chiba-0903.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • S03154 -> OK
  • R1,R3~R7 (Plac+RBS+RhlI+LVA) -> OK
Chiba-0903-2.JPG
From left;
  • C1,C2,C4~C8 (Plac+RBS+CinI+LVA) -> OK
--->Digestion test
  • R1,R3~R7 (Plac+RBS+RhlI+LVA)
  • C1,C2,C4~C8 (Plac+RBS+CinI+LVA)
Digestion SingleDouble
Sample DNA 13
XbaⅠ 0.10.1
PstⅠ 0.10.1
Buffer 2 0.9-
Buffer 3 --0.8
BSA 11
dH2O 75
TOTAL 1010


--->Gel Check
Chiba-0903-3.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
  • Single Digestion : R1,R3~R7 -> OK
  • Single Digestion : C1,C2,C4~C8 -> OK
Chiba-0903-4.JPG
From left;
  • Double Digestion : R1,R3~R7 -> OK
  • Double Digestion : C1,C2,C4~C8 -> OK


(2/9)--->Phenotype-test : mix

Sample No. 1234567891011
L1 2----------
L2-2---------
L3--2--------
L4---2-------
R1----1------
R2-----1-----
R3------1----
R4-------1---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------2--
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]22221111211
AHL11111111111
|
| 8h
V
Spindown
|
|
V
the intensity GFP

4 September, 2008

--->Gel Check
Chiba-0904.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
Chiba-0904-2.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;
Chiba-0904-3.JPG
Sample No ①②③④
Sample DNA 3333
Loading Dye 2222
dH2O 7777
TOTAL 12121212
From left;

5 September, 2008

6 September, 2008

>next week

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