Team:TzuChiU Formosa/Notebook/photopaper

From 2011.igem.org

(Difference between revisions)
Line 219: Line 219:
<br>
<br>
*[pSB1C3/PstⅠ]
*[pSB1C3/PstⅠ]
-
<br>
 
<br>DNA             500ng<br>
<br>DNA             500ng<br>
<br>10×buffer           5μl<br>
<br>10×buffer           5μl<br>
Line 248: Line 247:
<br>
<br>
*[pSB1C3/XbaⅠ+SpeⅠ]
*[pSB1C3/XbaⅠ+SpeⅠ]
-
<br>
 
<br>DNA               10μl<br>
<br>DNA               10μl<br>
<br>10×buffer            5μl<br>
<br>10×buffer            5μl<br>
Line 263: Line 261:
<br>
<br>
*[pSB1C3/SpeⅠ+PstⅠ]
*[pSB1C3/SpeⅠ+PstⅠ]
-
<br>
 
<br>DNA               10μl<br>
<br>DNA               10μl<br>
<br>10×buffer            5μl<br>
<br>10×buffer            5μl<br>
Line 324: Line 321:
<br>
<br>
*[pSB1C3/EcoRⅠ+PstⅠ]
*[pSB1C3/EcoRⅠ+PstⅠ]
-
<br>
 
<br>
<br>
*[pSB1C3/XbaⅠ+SpeⅠ]
*[pSB1C3/XbaⅠ+SpeⅠ]
-
<br>
 
<br>
<br>
*[pSB1C3/SpeⅠ+PstⅠ]
*[pSB1C3/SpeⅠ+PstⅠ]
-
<br>
 
<br>
<br>
*[pSB1C3/EcoRⅠ+XbaⅠ]
*[pSB1C3/EcoRⅠ+XbaⅠ]
-
<br>
 
<br>
<br>
*[pSB1A3/EcoRⅠ+PstⅠ]
*[pSB1A3/EcoRⅠ+PstⅠ]
-
<br>
 
<br>
<br>
*[pSB1A3/EcoRⅠ+SpeⅠ]
*[pSB1A3/EcoRⅠ+SpeⅠ]
Line 349: Line 341:
<br>
<br>
*[acsAB]  
*[acsAB]  
-
<br>
 
<br>
<br>
*[acsCD]  
*[acsCD]  
-
<br>
 
<br>
<br>
*[CCcax-Ccp]  
*[CCcax-Ccp]  
-
<br>
 
<br>template DNA   1μl<br>
<br>template DNA   1μl<br>
<br>5×Buffer     4μl<br>
<br>5×Buffer     4μl<br>
Line 374: Line 363:
*[acsAB]
*[acsAB]
<br>  
<br>  
-
<br>
 
*[acsCD]
*[acsCD]
<br>  
<br>  
-
<br>
 
*[CMCax-Ccp]  
*[CMCax-Ccp]  
<br>
<br>
Line 404: Line 391:
<br>
<br>
*[acsCD/XbaⅠ+SpeⅠ]
*[acsCD/XbaⅠ+SpeⅠ]
-
<br>
 
<br>DNA            10μl<br>
<br>DNA            10μl<br>
<br>10×buffer         5μl<br>
<br>10×buffer         5μl<br>
Line 452: Line 438:
<br>
<br>
*[acsAB/ XbaⅠ+SpeⅠ]
*[acsAB/ XbaⅠ+SpeⅠ]
-
<br>
 
<br>
<br>
*[acsCD/XbaⅠ+SpeⅠ]
*[acsCD/XbaⅠ+SpeⅠ]
-
<br>
 
<br>
<br>
*[CMCax/SpeⅠ+AlwNⅠ]
*[CMCax/SpeⅠ+AlwNⅠ]
-
<br>
 
<br>
<br>
*[Ccp/AlwNⅠ+PstⅠ]
*[Ccp/AlwNⅠ+PstⅠ]
Line 470: Line 453:
<br>
<br>
*[pSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]]
*[pSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 484: Line 466:
<br>
<br>
*[pSB1A3-acsCD]
*[pSB1A3-acsCD]
-
<br>
 
<br>Vector           3μl<br>
<br>Vector           3μl<br>
<br>Insert           14μl<br>
<br>Insert           14μl<br>
Line 498: Line 479:
<br>
<br>
*[pSB1C3-acsCD]
*[pSB1C3-acsCD]
-
<br>
 
<br>Vector           3μl<br>
<br>Vector           3μl<br>
<br>Insert           14μl<br>
<br>Insert           14μl<br>
Line 512: Line 492:
<br>
<br>
*[pSB1C3-CMCax-Ccp]
*[pSB1C3-CMCax-Ccp]
-
<br>
 
<br>Vector           3μl<br>
<br>Vector           3μl<br>
<br>Insert           14μl<br>
<br>Insert           14μl<br>
Line 532: Line 511:
<br>
<br>
*[R0011 promoter]
*[R0011 promoter]
-
<br>
 
<br>R0011 promoter         1μl<br>
<br>R0011 promoter         1μl<br>
<br>5×Buffer            4μl<br>
<br>5×Buffer            4μl<br>
Line 547: Line 525:
<br>
<br>
*[R0011 promoter]
*[R0011 promoter]
-
<br>
 
-
 
<br>
<br>
<br><font color="#000000" size=2><b>Transformation of DNA</b></font>
<br><font color="#000000" size=2><b>Transformation of DNA</b></font>
Line 554: Line 530:
<br>
<br>
*[pSB1C3-acsAB]
*[pSB1C3-acsAB]
-
<br>
 
<br>Transform into E.coli
<br>Transform into E.coli
<br>LB+CHLORAMPHENICOL
<br>LB+CHLORAMPHENICOL
Line 562: Line 537:
<br>
<br>
*[pSB1C3-acsCD]
*[pSB1C3-acsCD]
-
<br>
 
<br>Transform into E.coli
<br>Transform into E.coli
<br>LB+CHLORAMPHENICOL
<br>LB+CHLORAMPHENICOL
Line 569: Line 543:
<br>
<br>
*[pSB1A3-acsCD]
*[pSB1A3-acsCD]
-
<br>
 
<br>Transform into E.coli
<br>Transform into E.coli
<br>LB+Ampicillin
<br>LB+Ampicillin
Line 577: Line 550:
<br>
<br>
*[pSB1C3-CMCax-Ccp]
*[pSB1C3-CMCax-Ccp]
-
<br>
 
<br>Transform into E.coli
<br>Transform into E.coli
<br>LB+CHLORAMPHENICOL
<br>LB+CHLORAMPHENICOL
Line 588: Line 560:
<br>
<br>
*[R0011 promoter/EcoRⅠ+XbaⅠ]  
*[R0011 promoter/EcoRⅠ+XbaⅠ]  
-
<br>
 
<br>DNA           10μl<br>
<br>DNA           10μl<br>
<br>10×buffer        5μl<br>
<br>10×buffer        5μl<br>
Line 606: Line 577:
*[R0011 promoter/EcoRⅠ+XbaⅠ]  
*[R0011 promoter/EcoRⅠ+XbaⅠ]  
<br>
<br>
-
<br>
 
-
<br>
 
-
 
<br><font color="#000000" size=3><b>2011.09.24</b></font>
<br><font color="#000000" size=3><b>2011.09.24</b></font>
<br>
<br>
Line 615: Line 583:
<br>
<br>
*[pSB1C3-R0011]
*[pSB1C3-R0011]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 629: Line 596:
<br>
<br>
*[R0011-acsAB]
*[R0011-acsAB]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 642: Line 608:
<br>
<br>
*[R0011-acsCD]
*[R0011-acsCD]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 662: Line 627:
<br>
<br>
*[R0011-acsAB/EcoRⅠ+SpeⅠ]  
*[R0011-acsAB/EcoRⅠ+SpeⅠ]  
-
<br>
 
<br>DNA           10μl<br>
<br>DNA           10μl<br>
<br>10×buffer        5μl<br>
<br>10×buffer        5μl<br>
Line 676: Line 640:
<br>
<br>
*[R0011-acsCD/EcoRⅠ+SpeⅠ]  
*[R0011-acsCD/EcoRⅠ+SpeⅠ]  
-
<br>
 
<br>DNA           10μl<br>
<br>DNA           10μl<br>
<br>10×buffer        5μl<br>
<br>10×buffer        5μl<br>
Line 694: Line 657:
<br>
<br>
*[R0011-acsAB/EcoRⅠ+SpeⅠ]
*[R0011-acsAB/EcoRⅠ+SpeⅠ]
-
<br>
 
<br>
<br>
*[R0011-acsCD/EcoRⅠ+SpeⅠ]  
*[R0011-acsCD/EcoRⅠ+SpeⅠ]  
Line 707: Line 669:
<br>
<br>
*[pSB1C3-R0011-acsAB]
*[pSB1C3-R0011-acsAB]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 721: Line 682:
<br>
<br>
*[pSB1C3-R0011-acsCD]
*[pSB1C3-R0011-acsCD]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 740: Line 700:
<br>
<br>
*[pSB1C3-R0011-acsAB]
*[pSB1C3-R0011-acsAB]
-
<br>
 
<br>Transform into E.coli
<br>Transform into E.coli
<br>LB+CHLORAMPHENICOL
<br>LB+CHLORAMPHENICOL
Line 748: Line 707:
<br>
<br>
*[pSB1C3-R0011-acsCD]
*[pSB1C3-R0011-acsCD]
-
<br>
 
<br>Transform into E.coli
<br>Transform into E.coli
<br>LB+CHLORAMPHENICOL
<br>LB+CHLORAMPHENICOL
Line 758: Line 716:
<br>
<br>
*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]  
*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]  
-
<br>
 
<br>DNA           10μl<br>
<br>DNA           10μl<br>
<br>10×buffer        5μl<br>
<br>10×buffer        5μl<br>
Line 775: Line 732:
<br>
<br>
*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]  
*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]  
-
<br>
 
<br>
<br>
<br>
<br>
Line 785: Line 741:
<br>
<br>
*[pSB1C3-R0011-acsCD-CMCax-Ccp]
*[pSB1C3-R0011-acsCD-CMCax-Ccp]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>
Line 801: Line 756:
<br>
<br>
*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]  
*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]  
-
<br>
 
<br>DNA           10μl<br>
<br>DNA           10μl<br>
<br>10×buffer        5μl<br>
<br>10×buffer        5μl<br>
Line 818: Line 772:
<br>
<br>
*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]  
*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]  
-
<br>
 
<br>
<br>
<br>
<br>
Line 828: Line 781:
<br>
<br>
*[pSB1A3-R0011-acsCD-CMCax-Ccp]
*[pSB1A3-R0011-acsCD-CMCax-Ccp]
-
<br>
 
<br>Vector          3μl<br>
<br>Vector          3μl<br>
<br>Insert          14μl<br>
<br>Insert          14μl<br>

Revision as of 07:55, 4 October 2011

Photopaper

Meeting Notes


2011.02.24
Discussion:

  • Team organization
    caption
  • Brain storming
    • paper made by bacteria with add-ons such as colors, fragrance, etc.
    • "light up" the plants for replacing lamp posts.



2011.03.04
Discussion:

  • Team advisory
  • Brain storming
    • Add-ons: colorful cellulose which produced by bacteria such as beta-carotene
    • information exchange with iGEM 2009 Cambridge team




2011.03.14
Discussion:

  • Task Allocation
  • Brain storming
    • Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene
    • Eco-friendly warmer - biotic thermal pad



2011.03.23
Discussion:

    • Project : paperia
    • Option 1 : Culture bacteria which has pigment gene
    • Option 2 : Cellulose-producing bacteria secrete pigment into the medium



2011.03.24
Discussion:

  • Exp. procedure:
    • cloning of cellulose gene’s CDS
    • the product should operate within E. coli.



2011.06.22
Discussion:

  • Due to some unforseen reason, the team decided to change their project.
  • New project: Biojenny

         -economical and humane way to produce paper in large quantities.
         -yeast to be our host



2011.07.01
Discussion:

  • Freeze > grin > genome DNA isolation > Cloning = silk protein gene



2011.07.09
Discussion:

  • the connections between 3 silk proteins : Fibl Fibh P25
  • major proteins : H-chain, L-chain, P25



2011.07.15
Discussion:

  • Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !
  • However it would be modified to be more innovative and creative.



2011.07.18
Discussion:

  • Latest project : Photo paper
  • cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.



2011.07.23
Discussion:

  • system modification to overcome the problems arises during preliminary round
  • Biobricks from Tokyo 2010 team will be utilized
    • regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria




2011.09.15

Genome miniprep
Gluconacetobacter hansenii



2011.09.18

Gel/PCR DNA extraction
Gluconacetobacter hansenii


Protocols


2011.09.07


Rhodobacter rudrum medium

K2HPO4             1g

NaCl              0.5g

FeSO4.7H2O          0.01g

CaCl2              0.02g

MnCl2.4H2O          0.002g

MgSO4.7H2O          0.2g

NaMO2O4.2H2O         0.01g

ddH2O              998.258ml

________________________________________
                 1L →take100ml

          + 


Yeast Extrat                0.5g

Sodium malate
(Sodium succinate dibasic hexohydrate)   5g

NH4Cl                   1g

ddH2O                  893.5ml

_________________________________________________
                     1L



Raise E. coli(PSB1C3)

1.50ml LB+500μl CHLORAMPHENICOL
2.37℃, overnight (14-16hrs)



2011.09.08-13

Plasmid miniprep kit


PSB1C3 plasmid
caption




Raise Rhodobacter rubrum

1.50ml LB+500μl CHLORAMPHENICOL
2.37℃, overnight (14-16hrs)



2011.09.09

Raise Gluconacetobacter hansenii

1.50ml LB+500μl CHLORAMPHENICOL
2.37℃, overnight (14-16hrs)



2011.09.10-11

Digestion check of DNA

  • [pSB1C3/EcoRI]



DNA            500ng

10×buffer        5μl

BSA            5μl

EcoRⅠ           1μl

ddH2O          29μl

_______________________________

total           50μl



  • [pSB1C3/PstⅠ]


DNA            500ng

10×buffer           5μl

BSA              5μl

PstⅠ             1μl

ddH2O            29μl

_________________________________

total            50μl



Digestion of DNA

[pSB1C3/EcoRⅠ+PstⅠ]

DNA             500ng

10×buffer           5μl

BSA              5μl

EcoRⅠ             1μl

pstⅠ              1μl

ddH2O            28μl

__________________________________

total            50μl

→37℃ for 30 mins


  • [pSB1C3/XbaⅠ+SpeⅠ]


DNA               10μl

10×buffer            5μl

BSA               5μl

XbaⅠ               1μl

SpeⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


  • [pSB1C3/SpeⅠ+PstⅠ]


DNA               10μl

10×buffer            5μl

BSA               5μl

SpeⅠ              1μl

pstⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


  • [pSB1C3/EcoRⅠ+XbaⅠ]



DNA               10μl

10×buffer            5μl

BSA               5μl

EcoRⅠ              1μl

XbaⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


  • [pSB1A3/EcoRⅠ+PstⅠ]



DNA               10μl

10×buffer            5μl

BSA               5μl

EcoRⅠ              1μl

PstⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs

  • [pSB1A3/EcoRⅠ+SpeⅠ]



DNA               10μl

10×buffer            5μl

BSA               5μl

EcoRⅠ              1μl

SpeⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


electroelution Purification

  • [pSB1C3/EcoRⅠ+PstⅠ]


  • [pSB1C3/XbaⅠ+SpeⅠ]


  • [pSB1C3/SpeⅠ+PstⅠ]


  • [pSB1C3/EcoRⅠ+XbaⅠ]


  • [pSB1A3/EcoRⅠ+PstⅠ]


  • [pSB1A3/EcoRⅠ+SpeⅠ]





2011.09.12-20

PCR


  • [acsAB]


  • [acsCD]


  • [CCcax-Ccp]


template DNA   1μl

5×Buffer     4μl

2.5μM dNTP    1.6μl

10μM F      1μl

10μM R      1μl

Taq        0.2μl

ddH2O      8.8μl

_______________________________

total       20μl




electroelution Purification

  • [acsAB]


  • [acsCD]


  • [CMCax-Ccp]





2011.09.21

Digestion of DNA

  • [acsAB/ XbaⅠ+SpeⅠ]



DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

pstⅠ           1μl

ddH2O          28μl

____________________________

total             50μl

→37℃ for 16 hr


  • [acsCD/XbaⅠ+SpeⅠ]


DNA            10μl

10×buffer         5μl

BSA             5μl

XbaⅠ            1μl

SpeⅠ             1μl

ddH2O            28μl

__________________________________
total               50μl

→37℃ for 16 hr


  • [CMCax/SpeⅠ+AlwNⅠ]



DNA            10μl

10×buffer         5μl

BSA             5μl

SpeⅠ            1μl

AlwNⅠ             1μl

ddH2O            28μl

__________________________________

total               50μl

→37℃ for 16 hr


  • [Ccp/AlwNⅠ+PstⅠ]



DNA            10μl

10×buffer         5μl

BSA             5μl

AlwNⅠ            1μl

PstⅠ             1μl

ddH2O            28μl

__________________________________
total               50μl

→37℃ for 16 hr



electroelution Purification

  • [acsAB/ XbaⅠ+SpeⅠ]


  • [acsCD/XbaⅠ+SpeⅠ]


  • [CMCax/SpeⅠ+AlwNⅠ]


  • [Ccp/AlwNⅠ+PstⅠ]





2011.09.22

Ligation of DNA

  • [pSB1C3-acsAB]
    caption


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


  • [pSB1A3-acsCD]


Vector           3μl

Insert           14μl

ligase buffer       2μl

ligase            1μl

ddH2O             -μl

_________________________________

total           20μl

→16℃ for 16 hr


  • [pSB1C3-acsCD]


Vector           3μl

Insert           14μl

ligase buffer       2μl

ligase            1μl

ddH2O             -μl

_________________________________

total           20μl

→16℃ for 16 hr


  • [pSB1C3-CMCax-Ccp]


Vector           3μl

Insert           14μl

ligase buffer       2μl

ligase            1μl

ddH2O             -μl

_________________________________

total           20μl

→16℃ for 16 hr



2011.09.23

PCR

  • [R0011 promoter]


R0011 promoter        1μl

5×Buffer            4μl

2.5μM dNTP          1.6μl

Taq               0.2μl

ddH2O             13.2μl

_____________________________________

total              20μl



electroelution Purification

  • [R0011 promoter]



Transformation of DNA

  • [pSB1C3-acsAB]


Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr


  • [pSB1C3-acsCD]


Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr

  • [pSB1A3-acsCD]


Transform into E.coli
LB+Ampicillin
→37℃ for 14 hr


  • [pSB1C3-CMCax-Ccp]


Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr



Digestion of DNA

  • [R0011 promoter/EcoRⅠ+XbaⅠ]


DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

XbaⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr


electroelution Purification

  • [R0011 promoter/EcoRⅠ+XbaⅠ]



2011.09.24

Ligation of DNA

  • [pSB1C3-R0011]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


  • [R0011-acsAB]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr

  • [R0011-acsCD]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr



2011.09.24

Digestion of DNA

  • [R0011-acsAB/EcoRⅠ+SpeⅠ]


DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

SpeⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr

  • [R0011-acsCD/EcoRⅠ+SpeⅠ]


DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

SpeⅠ           1μl

ddH2O          28μl

____________________________

total             50μl

→37℃ for 16 hr



electroelution Purification

  • [R0011-acsAB/EcoRⅠ+SpeⅠ]


  • [R0011-acsCD/EcoRⅠ+SpeⅠ]





2011.09.25

Ligation of DNA

  • [pSB1C3-R0011-acsAB]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


  • [pSB1C3-R0011-acsCD]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


2011.09.26

Transformation of DNA

  • [pSB1C3-R0011-acsAB]


Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr


  • [pSB1C3-R0011-acsCD]


Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr


Digestion of DNA

  • [pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]


DNA           10μl

10×buffer        5μl

BSA           5μl

SpeⅠ          1μl

PstⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr


electroelution Purification

  • [pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]




2011.09.27

Ligation of DNA

  • [pSB1C3-R0011-acsCD-CMCax-Ccp]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


Digestion of DNA

  • [pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]


DNA           10μl

10×buffer        5μl

BSA           5μl

PstⅠ          1μl

EcoRⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr


electroelution Purification

  • [pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]




2011.09.28

Ligation of DNA

  • [pSB1A3-R0011-acsCD-CMCax-Ccp]


Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


2011.09.29

Transformation of DNA

  • [pSB1C3-R0011-acsAB]


  • [pSB1A3-R0011-acsAB-CMCax-Ccp]



Transform into E.coli
LB+Ampiclin+CHLORAMPHENICOL
→37℃ for 14 hr