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Team:HokkaidoU Japan/Notebook/Pre experiment/mRFP-expression-construct
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<div class="step-recipe"> pSB6A1 | <div class="step-recipe"> pSB6A1 | ||
| - | 1µL | + | 1µL to JM109</div> |
</div> | </div> | ||
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</ul> | </ul> | ||
<div class="step-recipe"> R0040-B0034-E1010-B0015 from our stock | <div class="step-recipe"> R0040-B0034-E1010-B0015 from our stock | ||
| - | 100up_F 200dn_R</div> | + | 100up_F Tm:70.4℃ & 200dn_R Tm:66.6℃ |
| + | </div> | ||
| + | <div class="step-recipe"> | ||
| + | KOD plus Neo</div> | ||
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<div class="step-recipe"> Cut R0040-B0034-E1010-B0015 with EcoRI, PstI (using 10×H buffer) | <div class="step-recipe"> Cut R0040-B0034-E1010-B0015 with EcoRI, PstI (using 10×H buffer) | ||
| - | Cut pSB6A1 with EcoRI, PstI (using 10×H buffer)</div> | + | </div> |
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| + | Cut pSB6A1 with EcoRI, PstI (using 10×H buffer) | ||
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<div class="step-recipe"> R0040-B0034-E1010-B0015 | <div class="step-recipe"> R0040-B0034-E1010-B0015 | ||
| - | pSB6A1</div> | + | pSB6A1 |
| + | </div> | ||
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| - | pSB6A1</div> | + | pSB6A1 |
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| - | <div class="step-recipe"> Ligate R0040-B0034-E1010-B0015 with pSB6A1</div> | + | <div class="step-recipe"> Ligate R0040-B0034-E1010-B0015 with pSB6A1 |
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| - | <div class="step-recipe"> R0040-B0034-E1010-B0015 on pSB6A1 | + | <div class="step-recipe"> R0040-B0034-E1010-B0015(on pSB6A1) |
| - | 5µL | + | 5µL to DH5α Turbo |
| + | </div> | ||
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| - | <div class="step-recipe"> R0040-B0034-E1010-B0015 on pSB6A1</div> | + | <div class="step-recipe"> R0040-B0034-E1010-B0015(on pSB6A1) |
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| - | <div class="step-recipe"> R0040-B0034-E1010-B0015 on pSB6A1</div> | + | <div class="step-recipe">R0040-B0034-E1010-B0015(on pSB6A1)</div> |
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Latest revision as of 15:24, 9 September 2015
Notebook
Lab Documents
mRFP Expression Plasmid Construction
-
Start
-
Get genes
pSB6A1 -
Transformation
pSB6A1 1µL to JM109 -
Liquid Culture
pSB6A1 -
Mini-prep
pSB6A1 -
PCR & PCR purification
R0040-B0034-E1010-B0015 from our stock 100up_F Tm:70.4℃ & 200dn_R Tm:66.6℃KOD plus Neo -
Digestion
Cut R0040-B0034-E1010-B0015 with EcoRI, PstI (using 10×H buffer)Cut pSB6A1 with EcoRI, PstI (using 10×H buffer) -
Gel Extraction
R0040-B0034-E1010-B0015 pSB6A1 -
Ethanol precipetation
R0040-B0034-E1010-B0015 pSB6A1 -
Dephosphorylation
R0040-B0034-E1010-B0015 -
Ligation
Ligate R0040-B0034-E1010-B0015 with pSB6A1 -
Transformation
R0040-B0034-E1010-B0015(on pSB6A1) 5µL to DH5α Turbo -
Liquid Culture
R0040-B0034-E1010-B0015(on pSB6A1) -
Mini-prep
R0040-B0034-E1010-B0015(on pSB6A1) -
Complete!
