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Team:Elan Vital South Korea/l miniprep
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<h1 class="title">Miniprep</h1> | <h1 class="title">Miniprep</h1> | ||
| - | <p class="listing">a. Transfer a single colony from the transformed E.colli to the LB | + | <p class="listing">a. Transfer a single colony from the transformed E.colli to the LB liquid broth and incubate in shaking incubator for 24 hours.</p> |
| - | <p class="listing">b. Centrifuge said E.colli for 15 minutes at | + | <p class="listing">b. Centrifuge said E.colli for 15 minutes at 1,500 rpm.</p> |
<p class="listing">c. Dispose of supernatant.</p> | <p class="listing">c. Dispose of supernatant.</p> | ||
<p class="listing">d. Add 300μl of solution I<i class="green">*</i> and vortex.</p> | <p class="listing">d. Add 300μl of solution I<i class="green">*</i> and vortex.</p> | ||
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<p class="listing">f. Add 300μl of solution II<i class="green">**</i> in each EP tube.</p> | <p class="listing">f. Add 300μl of solution II<i class="green">**</i> in each EP tube.</p> | ||
<p class="listing">g. Add 200μl of solution III<i class="green">***</i> in each EP tube and mix by inverting.</p> | <p class="listing">g. Add 200μl of solution III<i class="green">***</i> in each EP tube and mix by inverting.</p> | ||
| - | <p class="listing">h. Centrifuge both EP tubes for 10 minutes at | + | <p class="listing">h. Centrifuge both EP tubes for 10 minutes at 14,000rpm</p> |
<p class="listing">i. Transfer 550μl of supernatant to column. </p> | <p class="listing">i. Transfer 550μl of supernatant to column. </p> | ||
| - | <p class="listing">j. Centrifuge at | + | <p class="listing">j. Centrifuge at 14,000rpm for 1 minute.</p> |
<p class="listing">k. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.</p> | <p class="listing">k. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.</p> | ||
| - | <p class="listing">l. Centrifuge at | + | <p class="listing">l. Centrifuge at 14,000rpm for 1 minute.</p> |
<p class="listing">m. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.</p> | <p class="listing">m. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.</p> | ||
| - | <p class="listing">n. Centrifuge at | + | <p class="listing">n. Centrifuge at 14,000rpm for 1 minute.</p> |
<p class="listing">o. Dispose of fluid in collection tube.</p> | <p class="listing">o. Dispose of fluid in collection tube.</p> | ||
| - | <p class="listing">p. Centrifuge at | + | <p class="listing">p. Centrifuge at 14,000rpm for 1 minute.</p> |
| - | <p class="listing">q. Add 50μl of distilled water and centrifuge at | + | <p class="listing">q. Add 50μl of distilled water and centrifuge at 14,000rpm for 1 minute.</p> |
<p class="listing">r. Keep filtered solution.</p> | <p class="listing">r. Keep filtered solution.</p> | ||
<p class="listing_sub green">* solution Ⅰ 50mM glucose, 25mM Tris•Cl (pH 8.0), 10mM EDTA (pH 8.0)</p> | <p class="listing_sub green">* solution Ⅰ 50mM glucose, 25mM Tris•Cl (pH 8.0), 10mM EDTA (pH 8.0)</p> | ||
Latest revision as of 20:05, 20 June 2014
Miniprep
a. Transfer a single colony from the transformed E.colli to the LB liquid broth and incubate in shaking incubator for 24 hours.
b. Centrifuge said E.colli for 15 minutes at 1,500 rpm.
c. Dispose of supernatant.
d. Add 300μl of solution I* and vortex.
e. Transfer the fluid into 2 EP tubes, 150μl each.
f. Add 300μl of solution II** in each EP tube.
g. Add 200μl of solution III*** in each EP tube and mix by inverting.
h. Centrifuge both EP tubes for 10 minutes at 14,000rpm
i. Transfer 550μl of supernatant to column.
j. Centrifuge at 14,000rpm for 1 minute.
k. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.
l. Centrifuge at 14,000rpm for 1 minute.
m. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.
n. Centrifuge at 14,000rpm for 1 minute.
o. Dispose of fluid in collection tube.
p. Centrifuge at 14,000rpm for 1 minute.
q. Add 50μl of distilled water and centrifuge at 14,000rpm for 1 minute.
r. Keep filtered solution.
* solution Ⅰ 50mM glucose, 25mM Tris•Cl (pH 8.0), 10mM EDTA (pH 8.0)
** solution Ⅱ 0.2N NaOH, 1% SDS
*** solution Ⅲ 5M potassium acetate, glacial acetic acid, H₂O
