Team:TMU-Tokyo/Notebook/Protocol/

From 2012.igem.org

(Difference between revisions)
Line 64: Line 64:
 +
<p class="description">
 +
<b>■Plasmid DNA Purification</b>; <A Href="http://www.qiagen.com/products/genomicdnastabilizationpurification/generationcapturecolumnkit.aspx"></A> (QIAGEN)<Br></p>
 +
</p>
 +
<p class="description3">
 +
All centrifuge is done at 13,000rpm, room temperature.<Br>
 +
<Br>
 +
<b>1.</b> Add 1mL overnight cultures of <i>E. coli</i> in LB medium to eppendorf tube. Centrifuge 1 min and create pellet.<Br>
 +
<b>2.</b> Add 250µL Buffer P1 and vortex until pellet perfectly dissolved.<Br>
 +
<b>3.</b> Add 250µL Buffer P2 and mix softly by inverting the tube 4-6 times. (Don't vortex.)<Br>
 +
<b>4.</b> Add 350µL Buffer N3 and immediately mix softly by inverting the tube 4-6 times. (Don't vortex.)<Br>
 +
<b>5.</b> Centrifuge 10 min. Afterwards, add supernatant to <Br>
 +
<b>6.</b> <Br>
 +
<b>7.</b> <Br>
 +
<b>8.</b> <Br>
 +
<b>9.</b> <Br>
 +
<b>10.</b> <Br>
-
<b>■Plasmid DNA Purification</b><Br></p>
 
<Br>
<Br>
<Br>
<Br>
Line 74: Line 89:
<p class="description3">
<p class="description3">
<Br>
<Br>
-
<b>1.</b> Add 200µL overnight cultures of E. coli in LB medium to the Capture Column.<Br>
+
<b>1.</b> Add 200µL overnight cultures of <i>E. coli</i> in LB medium to the Capture Column.<Br>
<b>2.</b> Incubate 1 min at room temperature.<Br>
<b>2.</b> Incubate 1 min at room temperature.<Br>
<b>3.</b> Add 400µL DNA Purification Solution 1, incubate 1 min at room temperature.<Br>
<b>3.</b> Add 400µL DNA Purification Solution 1, incubate 1 min at room temperature.<Br>

Revision as of 13:29, 24 September 2012

 




■Experiment
1st week (8.13 - 8.19)
2nd week (8.20 - 8.26)
3rd week (8.27 - 9. 2)
4th week (9. 3 - 9. 9)
5th week (9.10 - 9.16)
6th week (9.17 - 9.23)
7th week (9.24 - 9.30)


■Protocols
Plasmid DNA Purification
Genome DNA Purification
Restruction Enzyme Degestion
DNA Fragment Ligation
Transformation
Electrophoresis
LB Medium


■Assay
Device1 Assay
Device2 Assay
Device3 Assay




Protocol



Our experiment was done follow description.


■Plasmid DNA Purification; (QIAGEN)

All centrifuge is done at 13,000rpm, room temperature.

1. Add 1mL overnight cultures of E. coli in LB medium to eppendorf tube. Centrifuge 1 min and create pellet.
2. Add 250µL Buffer P1 and vortex until pellet perfectly dissolved.
3. Add 250µL Buffer P2 and mix softly by inverting the tube 4-6 times. (Don't vortex.)
4. Add 350µL Buffer N3 and immediately mix softly by inverting the tube 4-6 times. (Don't vortex.)
5. Centrifuge 10 min. Afterwards, add supernatant to
6.
7.
8.
9.
10.


■Genome DNA Purification; Generation™ Capture Column (QIAGEN)


1. Add 200µL overnight cultures of E. coli in LB medium to the Capture Column.
2. Incubate 1 min at room temperature.
3. Add 400µL DNA Purification Solution 1, incubate 1 min at room temperature.
4. Centrifuge 10 s with tabletop centrifuge.
5. Transfer the Capture Column to the Waste Collection Tube.
6. Add 400µL DNA Purification Solution 1, incubate 1 min at room temperature.
7. Centrifuge 10 s with tabletop centrifuge.
8. Add 200µl DNA Elution Solution 2. (Don't incubate.)
9. Centrifuge 10 s with tabletop centrifuge.
10. Transfer the Capture Column to DNA Collection Tube.
11. Add 100µl DNA Elution Solution 2, incuvate 10 min at 99℃ with heat block.
12. Centrifuge 20 s with tabletop centrifuge. Purified DNA will be released to solution.



■Restruction Enzyme Degestion



■DNA Fragment Ligation



■Transformation



■Electrophoresis



■LB Medium



■PCR


・PCR; PrimeSTAR® HS DNA Polymerase

・Colony PCR ; Takara EX®Taq