Team:Chiba/jk/β/week 3

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>Protocols


Contents

Week 3

>last week

31 August, 2008

Transformation
Competent Cells : XL10G
  • BBa_K084007(Plac+RBS+LasI)[http://partsregistry.org/Part:BBa_K084007]
  • BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008]
---> 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


--->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[http://partsregistry.org/Part:BBa_S03154]-->

(2/9)Mini prepwith BBa_K084009[http://partsregistry.org/Part:BBa_K084009], BBa_K084010[http://partsregistry.org/Part:BBa_K084010]

  • 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)[http://partsregistry.org/Part:BBa_K084009] -> 628 colonies
  2. BBa_K084010(Plac+RBS+CinI+LVA, Amp)[http://partsregistry.org/Part:BBa_K084010] -> 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)[http://partsregistry.org/Part:BBa_K084007]
  • BBa_K084008(Plac+RBS+RhlI)[http://partsregistry.org/Part:BBa_K084008]
  • BBa_T9002(Ptet+RBS+LuxR+GFP)[http://partsregistry.org/Part:BBa_K084010]
--->(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[http://partsregistry.org/Part:BBa_K084009](R1~R8),(2)BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C1~C8)) 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)--->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)[http://partsregistry.org/Part:BBa_K084007]
  • BBa_K084008(Plac+RBS+RhlI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_K084008]
  • BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_T9002]
from Glycerol Stock:
  • BBa_S03623(Ptet+RBS+LuxI, Competent Cells : BW ΔFliC)[http://partsregistry.org/Part:BBa_S03623]

--->(3/9)Phenotype test


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
  • BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R1, R3~R7)
  • BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C1,C2,C4~C8)
  • BBa_S03154[http://partsregistry.org/Part:BBa_S03154]


--->Gel Check
Chiba-0903.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
  • S03154[http://partsregistry.org/Part:BBa_S03154] -> OK 33ng/μL
  • BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R1, R3~R7) -> OK
Chiba-0903-2.JPG
From left;
  • BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C1,C2,C4~C8) -> OK
--->Digestion test
  • BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R1, R3~R7)
  • BBa_K084010[http://partsregistry.org/Part:BBa_K084010](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 10μL10μL


--->Gel Check
Chiba-0903-3.JPG
Sample DNA 10
Loading Dye 2
TOTAL 12
From left;
  • Single Digestion : BBa_K084009[http://partsregistry.org/Part:BBa_K084009]R1,R3~R7 -> OK
  • Single Digestion : BBa_K084010[http://partsregistry.org/Part:BBa_K084010]C1,C2,C4~C8 -> OK
Chiba-0903-4.JPG
From left;
  • Double Digestion : BBa_K084009[http://partsregistry.org/Part:BBa_K084009]R1,R3~R7 -> OK
  • Double Digestion : BBa_K084010[http://partsregistry.org/Part:BBa_K084010]C1,C2,C4~C8 -> OK


(2/9)--->Phenotype-test
  • MIX
  • BBa_K084007(Plac+RBS+LasI, Competent Cells : JW1908)[http://partsregistry.org/Part:BBa_K084007] -> Sample Name : L1~L4
  • BBa_K084008(Plac+RBS+RhlI, Competent Cells : JW1908)[http://partsregistry.org/Part:BBa_K084008] -> Sample Name : R1~R4
  • BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)[http://partsregistry.org/Part:BBa_S03623]
  • BBa_T9002(Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)[http://partsregistry.org/Part:BBa_T9002]


Sample No. 1234567891011
L1 2mL----------
L2-2mL---------
L3--2mL--------
L4---2mL-------
R1----1mL------
R2-----1mL-----
R3------1mL----
R4-------1mL---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------2mL--
AHL(100μM)----------1μL
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]2mL2mL2mL2mL1mL1mL1mL1mL2mL1mL1mL
IPTG(100μM)1μL1μL1μL1μL1μL1μL1μL1μL---
  • Incubated for 8hours at 37 degrees
  • Spindown (max rpm, 3 min)
  • The measurement of the intensity GFP(visual judgment)
Sample No 1234567891011
the intensity GFP +++++++++++++-


  • Leaving for 12 at room temperture.
  • Resuspension
  • The measurement of the intensity GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
Sample No 1234567891011
the intensity GFP 25.6518.9120.3921.8526.6226.5830.0733.6027.4110.7255.54

4 September, 2008

--->Gel Check
Chiba-0904.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μL
From left;
BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK
BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK
BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK
BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK
Chiba-0904-2.JPG
From left;
BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK
BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0161] -> OK
BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK
BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0161] -> OK


--->Digestion test
Sample Single DigesitonDouble Digestion
Sample DNA12
EcoRⅠ0.050.05
PstⅠ-0.05
Buffer 3 11
BSA 11
dH2O 6.955.9
TOTAL 10μL10μL


--->Gel Check
Chiba-0904-3.JPG


Sample DNA 10
Loading Dye 2
TOTAL 12μL
From left;
Single Digestion
  • BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261]
  • BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261]
  • BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261]
  • BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261]
ladder
Double digestion
  • BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0161(2007)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0161(2006)[http://partsregistry.org/Part:BBa_C0161]
  • BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261]
  • BBa_C0261(2007)[http://partsregistry.org/Part:BBa_C0261]
  • BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261]
  • BBa_C0261(2006)[http://partsregistry.org/Part:BBa_C0261]


Transformation
Competent cells : BW ΔFliC
Transformed the following and grew on new ampicillin plates.
  • BBa_K084009[http://partsregistry.org/Part:BBa_K084009](R-3,4,6,7)
  • BBa_K084010[http://partsregistry.org/Part:BBa_K084010](C-1,2,6,7)
  • BBa_T9002(2007)[http://partsregistry.org/Part:BBa_T9002]
--->(5/9)Liquid Culture

5 September, 2008

(4/9)--->Liquid Culture
Cultured the following cells (2mL LB-Amp, at 37℃)
from transformed plates:
  • BBa_K084009(Plac+RBS+RhlI+LVA, Competent Cells : JW1908) [http://partsregistry.org/Part:BBa_K084009](r1, r3~r7)
  • BBa_K084010(Plac+RBS+RhlI, Competent Cells : JW1908)[http://partsregistry.org/Part:BBa_K084010](c1,c2,c4~c8)
from Glycerol Stock:
  • BBa_S03623(Ptet+RBS+LuxI, Competent Cells : JW1908)[http://partsregistry.org/Part:BBa_S03623]
--->Phenotype test
  • MIX
Sample No. 1234567891011
r3 1mL----------
r4-1mL---------
r6--1mL--------
r7---1mL-------
c1----1mL------
c2-----1mL-----
c6------1mL----
c7-------1mL---
BBa_S03154[http://partsregistry.org/Part:BBa_S03154]--------1mL--
AHL(100μM)----------1μL
BBa_T9002[http://partsregistry.org/Part:BBa_T9002]1mL1mL1mL1mL1mL1mL1mL1mL1mL1mL1mL
IPTG(100μM)1μL1μL1μL1μL1μL1μL1μL1μL---


  • Incubated for 8hours at 37 degrees
  • The measurement of the intensity GFP by Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
Sample No 1234567891011
the intensity GFP (Frist run) 15.2116.4416.3416.639.7209.7039.6859.87419.9516.5971.04
the intensity GFP (Second run) 17.8519.1919.2819.149.98110.1610.1410.4123.9417.6179.53


  • Spindown (max rpm, 3 min)
  • The measurement of the intensity GFP(visual judgment) (UV 312nm)
Sample No 1234567891011
the intensity GFP ++++----+++-

6 September, 2008

Colony-PCR

PCR of BBa_I9030 and two control,BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 5
Reverse Primer 5
DNA polymerase 1
Thermopol Buffer 5
dH2O 33
TOTAL 50μL


95℃,5min -> ( 95℃,1min -> 54℃,1min -> 72℃,1min )･･･25cycles -> 72℃,10min -> 6℃ store


--->Gel Check
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6
From left;
1 I9030 without LVA -> OK
2 I9030 Positive Control -> OK
3 F2620 Positive control -> OK

Gel Check Sample DNA : R0010

Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6



Digestion Test

Sample DNA:

1:R0010,2:S03154,3:J04500,4:C0161,5:C0261

Sample No 12345
Sample DNA 11111
PstⅠ 0.50.50.50.50.5
SpeⅠ 0.5-1--
XbaⅠ -0.5-0.50.5
Buffer 2 2-3--
Buffer 3 -6-66
(10×)BSA 20.63--
(100×)BSA ---0.60.6
dH2O 36.471.41.4
TOTAL 2060306060

>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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