Team:Chiba/jk/β/week 3

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>送受信班

Contents

Week 3

>last week

31 August, 2008

Transformation
Competent Cells : XL10G
  • BBa_K084007(Plac+RBS+LasI)[1]
  • BBa_K084008(Plac+RBS+RhlI)[2]
---> We saved these plates with a refrigerator.


Digestion
  1. BBa_I9026[3](2007)
  2. BBa_I9030[4](2006)
  3. BBa_S03154[5](2007)
  4. BBa_R0010[6](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[7]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[8]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[9]①、②
  • Double Digestion Plac+RBS+RhlI 2~5
  • Double Digestion BBa_T9002[10]①、②
  • BBa_T9002[11]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


--->Gel Check
Chiba-0901-4.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • insert-1(I9026) -> OK
  • insert-2(I9030) -> OK
  • insert-3(S03154) -> None --> Transformation BBa_S03154[12]-->

(2/9)Mini prepwith BBa_K084009[13], BBa_K084010[14]

  • vector-4(R0010)


--->Ligation
Sample No (1)(2)(3)(4)(5)
insert-1(I9026) 3-3--
insert-2(I9030) -3-3-
vector-4(R0010) 33--3
ligase 11111
Buffer 11111
dH2O 22555
TOTAL 1010101010


--->Transformation
Competent cells : XL10GOLD 30μL
Transformed the following and grew on new ampicillin plates.
  1. BBa_K084009(Plac+RBS+RhlI+LVA, Amp)[15] -> 628 colonies
  2. BBa_K084010(Plac+RBS+CinI+LVA, Amp)[16] -> 500 colonies
  3. insert-1(RBS+RhlI+LVA) -> 9 colonies
  4. insert-2(RBS+CinI+LVA) -> No colonies on the plate
  5. vector-4(Plac, Amp) -> 186 colonies


--->(2/9) Colony PCR


Transformation

Competent cells : BW ΔFliC 40μL
Transformed the following and grew on new ampicillin plates.
  • BBa_K084007(Plac+RBS+LasI)[17]
  • BBa_K084008(Plac+RBS+RhlI)[18]
  • BBa_T9002(Ptet+RBS+LuxR+GFP)[19]
--->(2/9)Liquid Culture



2 September, 2008

(31/8)--->Gel Check
Chiba-0902.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
I9026 -> OK, 100/μL
I9030 -> OK, 50ng/μL
S03154 -> OK, 30ng/μL (too low for the ligation:1/9 )


(1/9)---> Colony PCR
Colony PCR of 8 colonies from ligation plates (1/9:(1)BBa_K084009[20](R1~R8),(2)BBa_K084010[21](C1~C8)) and one from control plate(BBa_F2620[22](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[23](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->Liquid Culture

Cultured the following cells (2mL LB-Amp, at 37℃, 7 hours)
from transformed plates:
  • BBa_K084007(Plac+RBS+LasI, Competent Cells : BW ΔFliC)[24]
  • BBa_K084008(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[25]
  • BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : BW ΔFliC)[26]
from Glycerol Stock:
  • BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)[27]

--->(3/9)Phenotype test


Transformation

Competent cells : XL10G 30μL

--->(4/9)Mini prep

3 September, 2008

(2/9)--->Mini prep
  • BBa_K084009[32](R1, R3~R7)
  • BBa_K084010[33](C1,C2,C4~C8)
  • BBa_S03154[34]


--->Gel Check
Chiba-0903.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • S03154[35] -> OK 33ng/μL
  • BBa_K084009[36](R1, R3~R7) -> OK
Chiba-0903-2.JPG
From left;
  • BBa_K084010[37](C1,C2,C4~C8) -> OK
--->Digestion test
  • BBa_K084009[38](R1, R3~R7)
  • BBa_K084010[39](C1,C2,C4~C8)
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 : BBa_K084009[40]R1,R3~R7 -> OK
  • Single Digestion : BBa_K084010[41]C1,C2,C4~C8 -> OK
Chiba-0903-4.JPG
From left;
  • Double Digestion : BBa_K084009[42]R1,R3~R7 -> OK
  • Double Digestion : BBa_K084010[43]C1,C2,C4~C8 -> OK


(2/9)--->Phenotype-test
  • MIX
  • BBa_K084007(Plac+RBS+LasI, Competent Cells : BW ΔFliC)[44] -> Sample Name : L1~L4
  • BBa_K084008(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[45] -> Sample Name : R1~R4
  • BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)[46]
  • BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : BW ΔFliC)[47]


Sample No. 1234567891011
L1 2----------
L2-2---------
L3--2--------
L4---2-------
R1----1------
R2-----1-----
R3------1----
R4-------1---
BBa_S03154[48]--------2--
BBa_T9002[49]22221111211
AHL(100μM)11111111111
  • Incubated for 8hours at 37 degrees
  • Spindown (max rpm, 3 min)
  • The measurement of the intensity GFP by Masahiro Tominaga
Sample No 1234567891011
the intensity GFP ++++++++++-+++

4 September, 2008

--->Gel Check
Chiba-0904.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
BBa_C0161(2007)[50] -> OK
BBa_C0161(2007)[51] -> OK
BBa_C0161(2006)[52] -> OK
BBa_C0161(2006)[53] -> OK
Chiba-0904-2.JPG
From left;
BBa_C0261(2007)[54] -> OK
BBa_C0261(2007)[55] -> OK
BBa_C0261(2006)[56] -> OK
BBa_C0261(2006)[57] -> OK


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


--->Gel Check
Chiba-0904-3.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
Single Digestion
  • BBa_C0161(2007)[58]
  • BBa_C0161(2007)[59]
  • BBa_C0161(2006)[60]
  • BBa_C0161(2006)[61]
  • BBa_C0261(2007)[62]
  • BBa_C0261(2007)[63]
  • BBa_C0261(2006)[64]
  • BBa_C0261(2006)[65]
ladder
Double digestion
  • BBa_C0161(2007)[66]
  • BBa_C0161(2007)[67]
  • BBa_C0161(2006)[68]
  • BBa_C0161(2006)[69]
  • BBa_C0261(2007)[70]
  • BBa_C0261(2007)[71]
  • BBa_C0261(2006)[72]
  • BBa_C0261(2006)[73]

5 September, 2008

6 September, 2008

>next week


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