"
Team:TzuChiU Formosa/Notebook/photopaper
From 2011.igem.org
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<br> | <br> | ||
<br> | <br> | ||
| - | <br><font color="#000000" size= | + | <br><font color="#000000" size=2><b>Raise Rhodobacter rubrum</b></font> |
<br> | <br> | ||
<br>1.50ml LB+500μl CHLORAMPHENICOL | <br>1.50ml LB+500μl CHLORAMPHENICOL | ||
| Line 204: | Line 204: | ||
<br><font color="#000000" size=2><b>Digestion check of DNA</b></font> | <br><font color="#000000" size=2><b>Digestion check of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3/EcoRI] | + | <br>*[pSB1C3/EcoRI] |
<br> | <br> | ||
<br>DNA 500ng<br> | <br>DNA 500ng<br> | ||
| Line 216: | Line 216: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3/PstⅠ] | + | <br>*[pSB1C3/PstⅠ] |
<br> | <br> | ||
<br>DNA 500ng<br> | <br>DNA 500ng<br> | ||
| Line 228: | Line 228: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br><font color="#000000" size= | + | <br><font color="#000000" size=2><b>Digestion of DNA</b></font> |
<br> | <br> | ||
<br>[pSB1C3/EcoRⅠ+PstⅠ] | <br>[pSB1C3/EcoRⅠ+PstⅠ] | ||
| Line 244: | Line 244: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3/XbaⅠ+SpeⅠ] | + | <br>*[pSB1C3/XbaⅠ+SpeⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 258: | Line 258: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3/SpeⅠ+PstⅠ] | + | <br>*[pSB1C3/SpeⅠ+PstⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 272: | Line 272: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3/EcoRⅠ+XbaⅠ] | + | <br>*[pSB1C3/EcoRⅠ+XbaⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 286: | Line 286: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1A3/EcoRⅠ+PstⅠ] | + | <br>*[pSB1A3/EcoRⅠ+PstⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 299: | Line 299: | ||
<br>→37℃ for 2 hrs | <br>→37℃ for 2 hrs | ||
<br> | <br> | ||
| - | <br>[pSB1A3/EcoRⅠ+SpeⅠ] | + | <br>*[pSB1A3/EcoRⅠ+SpeⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 315: | Line 315: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3/EcoRⅠ+PstⅠ] | + | <br>*[pSB1C3/EcoRⅠ+PstⅠ] |
<br> | <br> | ||
| - | <br>[pSB1C3/XbaⅠ+SpeⅠ] | + | <br>*[pSB1C3/XbaⅠ+SpeⅠ] |
<br> | <br> | ||
| - | <br>[pSB1C3/SpeⅠ+PstⅠ] | + | <br>*[pSB1C3/SpeⅠ+PstⅠ] |
<br> | <br> | ||
| - | <br>[pSB1C3/EcoRⅠ+XbaⅠ] | + | <br>*[pSB1C3/EcoRⅠ+XbaⅠ] |
<br> | <br> | ||
| - | <br>[pSB1A3/EcoRⅠ+PstⅠ] | + | <br>*[pSB1A3/EcoRⅠ+PstⅠ] |
<br> | <br> | ||
| - | <br>[pSB1A3/EcoRⅠ+SpeⅠ] | + | <br>*[pSB1A3/EcoRⅠ+SpeⅠ] |
<br> | <br> | ||
<br> | <br> | ||
| Line 334: | Line 334: | ||
<br><font color="#000000" size=2><b>PCR</b></font> | <br><font color="#000000" size=2><b>PCR</b></font> | ||
[[File:Cats2.gif |right|404px|caption]] | [[File:Cats2.gif |right|404px|caption]] | ||
| - | <br>[acsAB] | + | <br>*[acsAB] |
<br> | <br> | ||
| - | <br>[acsCD] | + | <br>*[acsCD] |
<br> | <br> | ||
| - | <br>[CCcax-Ccp] | + | <br>*[CCcax-Ccp] |
<br> | <br> | ||
<br>template DNA 1μl<br> | <br>template DNA 1μl<br> | ||
| Line 355: | Line 355: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[acsAB] | + | <br>*[acsAB] |
<br> | <br> | ||
| - | <br>[acsCD] | + | <br>*[acsCD] |
<br> | <br> | ||
| - | <br>[CMCax-Ccp] | + | <br>*[CMCax-Ccp] |
<br> | <br> | ||
<br> | <br> | ||
| Line 368: | Line 368: | ||
<br><font color="#000000" size=2><b>Digestion of DNA</b></font> | <br><font color="#000000" size=2><b>Digestion of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[acsAB/ XbaⅠ+SpeⅠ] | + | <br>*[acsAB/ XbaⅠ+SpeⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 382: | Line 382: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[acsCD/XbaⅠ+SpeⅠ] | + | <br>*[acsCD/XbaⅠ+SpeⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 396: | Line 396: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[CMCax/SpeⅠ+AlwNⅠ] | + | <br>*[CMCax/SpeⅠ+AlwNⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 410: | Line 410: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[Ccp/AlwNⅠ+PstⅠ] | + | <br>*[Ccp/AlwNⅠ+PstⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 427: | Line 427: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[acsAB/ XbaⅠ+SpeⅠ] | + | <br>*[acsAB/ XbaⅠ+SpeⅠ] |
<br> | <br> | ||
| - | <br>[acsCD/XbaⅠ+SpeⅠ] | + | <br>*[acsCD/XbaⅠ+SpeⅠ] |
<br> | <br> | ||
| - | <br>[CMCax/SpeⅠ+AlwNⅠ] | + | <br>*[CMCax/SpeⅠ+AlwNⅠ] |
<br> | <br> | ||
| - | <br>[Ccp/AlwNⅠ+PstⅠ] | + | <br>*[Ccp/AlwNⅠ+PstⅠ] |
<br> | <br> | ||
<br> | <br> | ||
| Line 441: | Line 441: | ||
<br> | <br> | ||
<br><font color="#000000" size=2><b>Ligation of DNA</b></font> | <br><font color="#000000" size=2><b>Ligation of DNA</b></font> | ||
| - | <br>[pSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]] | + | <br>*[pSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 454: | Line 454: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1A3-acsCD] | + | <br>*[pSB1A3-acsCD] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 467: | Line 467: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-acsCD] | + | <br>*[pSB1C3-acsCD] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 480: | Line 480: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-CMCax-Ccp] | + | <br>*[pSB1C3-CMCax-Ccp] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 499: | Line 499: | ||
<br><font color="#000000" size=2><b>PCR</b></font> | <br><font color="#000000" size=2><b>PCR</b></font> | ||
<br> | <br> | ||
| - | <br>[R0011 promoter] | + | <br>*[R0011 promoter] |
<br> | <br> | ||
<br>R0011 promoter 1μl<br> | <br>R0011 promoter 1μl<br> | ||
| Line 513: | Line 513: | ||
<br><font color="#000000" size=3><b>electroelution Purification</b></font> | <br><font color="#000000" size=3><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[R0011 promoter] | + | <br>*[R0011 promoter] |
<br> | <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> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-acsAB] | + | <br>*[pSB1C3-acsAB] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
| Line 525: | Line 525: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-acsCD] | + | <br>*[pSB1C3-acsCD] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
| Line 531: | Line 531: | ||
<br>→37℃ for 14 hr | <br>→37℃ for 14 hr | ||
<br> | <br> | ||
| - | <br>[pSB1A3-acsCD] | + | <br>*[pSB1A3-acsCD] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
| Line 538: | Line 538: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-CMCax-Ccp] | + | <br>*[pSB1C3-CMCax-Ccp] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
| Line 548: | Line 548: | ||
<br><font color="#000000" size=2><b>Digestion of DNA</b></font> | <br><font color="#000000" size=2><b>Digestion of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[R0011 promoter/EcoRⅠ+XbaⅠ] | + | <br>*[R0011 promoter/EcoRⅠ+XbaⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 564: | Line 564: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[R0011 promoter/EcoRⅠ+XbaⅠ] | + | <br>*[R0011 promoter/EcoRⅠ+XbaⅠ] |
<br> | <br> | ||
<br> | <br> | ||
| Line 573: | Line 573: | ||
<br><font color="#000000" size=2><b>Ligation of DNA</b></font> | <br><font color="#000000" size=2><b>Ligation of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011] | + | <br>*[pSB1C3-R0011] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 586: | Line 586: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[R0011-acsAB] | + | <br>*[R0011-acsAB] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 598: | Line 598: | ||
<br>→16℃ for 16 hr | <br>→16℃ for 16 hr | ||
<br> | <br> | ||
| - | <br>[R0011-acsCD] | + | <br>*[R0011-acsCD] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 617: | Line 617: | ||
<br><font color="#000000" size=2><b>Digestion of DNA</b></font> | <br><font color="#000000" size=2><b>Digestion of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[R0011-acsAB/EcoRⅠ+SpeⅠ] | + | <br>*[R0011-acsAB/EcoRⅠ+SpeⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 630: | Line 630: | ||
<br>→37℃ for 16 hr | <br>→37℃ for 16 hr | ||
<br> | <br> | ||
| - | <br>[R0011-acsCD/EcoRⅠ+SpeⅠ] | + | <br>*[R0011-acsCD/EcoRⅠ+SpeⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 647: | Line 647: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[R0011-acsAB/EcoRⅠ+SpeⅠ] | + | <br>*[R0011-acsAB/EcoRⅠ+SpeⅠ] |
<br> | <br> | ||
| - | <br>[R0011-acsCD/EcoRⅠ+SpeⅠ] | + | <br>*[R0011-acsCD/EcoRⅠ+SpeⅠ] |
<br> | <br> | ||
<br> | <br> | ||
| Line 658: | Line 658: | ||
<br><font color="#000000" size=2><b>Ligation of DNA</b></font> | <br><font color="#000000" size=2><b>Ligation of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsAB] | + | <br>*[pSB1C3-R0011-acsAB] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 671: | Line 671: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD] | + | <br>*[pSB1C3-R0011-acsCD] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 689: | Line 689: | ||
<br><font color="#000000" size=2><b>Transformation of DNA</b></font> | <br><font color="#000000" size=2><b>Transformation of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsAB] | + | <br>*[pSB1C3-R0011-acsAB] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
| Line 696: | Line 696: | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD] | + | <br>*[pSB1C3-R0011-acsCD] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
| Line 705: | Line 705: | ||
<br><font color="#000000" size=2><b>Digestion of DNA</b></font> | <br><font color="#000000" size=2><b>Digestion of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ] | + | <br>*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 721: | Line 721: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ] | + | <br>*[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ] |
<br> | <br> | ||
<br> | <br> | ||
| Line 730: | Line 730: | ||
<br><font color="#000000" size=2><b>Ligation of DNA</b></font> | <br><font color="#000000" size=2><b>Ligation of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD-CMCax-Ccp] | + | <br>*[pSB1C3-R0011-acsCD-CMCax-Ccp] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 745: | Line 745: | ||
<br><font color="#000000" size=2><b>Digestion of DNA</b></font> | <br><font color="#000000" size=2><b>Digestion of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ] | + | <br>*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ] |
<br> | <br> | ||
<br>DNA 10μl<br> | <br>DNA 10μl<br> | ||
| Line 761: | Line 761: | ||
<br><font color="#000000" size=2><b>electroelution Purification</b></font> | <br><font color="#000000" size=2><b>electroelution Purification</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ] | + | <br>*[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ] |
<br> | <br> | ||
<br> | <br> | ||
| Line 770: | Line 770: | ||
<br><font color="#000000" size=2><b>Ligation of DNA</b></font> | <br><font color="#000000" size=2><b>Ligation of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1A3-R0011-acsCD-CMCax-Ccp] | + | <br>*[pSB1A3-R0011-acsCD-CMCax-Ccp] |
<br> | <br> | ||
<br>Vector 3μl<br> | <br>Vector 3μl<br> | ||
| Line 787: | Line 787: | ||
<br><font color="#000000" size=2><b>Transformation of DNA</b></font> | <br><font color="#000000" size=2><b>Transformation of DNA</b></font> | ||
<br> | <br> | ||
| - | <br>[pSB1C3-R0011-acsAB] | + | <br>*[pSB1C3-R0011-acsAB] |
| - | <br>[pSB1A3-R0011-acsAB-CMCax-Ccp] | + | <br>*[pSB1A3-R0011-acsAB-CMCax-Ccp] |
<br> | <br> | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
Revision as of 07:04, 4 October 2011
Photopaper
Meeting Notes
2011.02.24
Discussion:
- Team organization
- 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
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]
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
