Team:Chiba/jk/β/weaks 3-4

From 2008.igem.org

Contents

Week 3

31 August, 2008

Transformation
Competent Cells : XL10G
  • Plac+RBS+LasI
  • Plac+RBS+RhlI

Digestion

  1. BBa_I9026[1](2007)
  2. BBa_I9030[2](2006)
  3. BBa_S03154[3](2007)
  4. BBa_R0010[4](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[5]①、②
Sample Single Digesiton 2~5Double Digestion 2~5Single Digestion T9002①Single Digesiton T9002②<Double 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[6]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[7]①、②
  • Double Digestion Plac+RBS+RhlI 2~5
  • Double Digestion BBa_T9002[8]①、②
  • BBa_T9002[9]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)
--->zymo
vector-4(R0010)
-> 15μL
--->SAP
vectr-4(R0010)
--->Zymo
1 -> 7μL
2 -> 7
3 -> 7
4 -> 20


Transformation

Competent cells : BW ΔFliC 40μL
  • Plac+RBS+LasI : Sample No.L1~L4
  • Plac+RBS+RhlI : 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[10](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[11](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->OD測


Transformation

Competent cells : XL10G 30μL
  • C0161(2007)
  • C0161(2006)
  • C0261(2007)
  • C0261(2006)
--->(4/9)miniprep

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[12]--------2--
BBa_T9002[13]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

Week 4

7 September, 2008

8 September, 2008

9 September, 2008

10 September, 2008

11 September, 2008

12 September, 2008

13 September, 2008

Gel Check
  1. BBa_C0161[14](2007)
  2. BBa_J04500[15](2007)
  3. BBa_R0010[16](2007)
Chiba-0913.JPG
Sample No 1~3
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;


--->[Team:Chiba/protocol/digestion|Digestion]]

  1. BBa_C0161[17](2007)
  2. BBa_J04500[18](2007)
  3. BBa_R0010[19](2007)


Sample No ①②③④
Sample DNA 5511
EcoRⅠ 0.5o.5--
SpeⅠ 0.50.5--
Buffer 1 11--
Buffer 3 --1-
BSA 11--
dH2O 227.5-
TOTAL 1010101
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