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Team:Elan Vital South Korea/l miniprep
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<p class="listing">b. Centrifuge said E.colli for 15 minutes at 1500 rpm.</p> | <p class="listing">b. Centrifuge said E.colli for 15 minutes at 1500 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 and vortex.</p> | + | <p class="listing">d. Add 300μl of solution I* and vortex.</p> |
<p class="listing">e. Transfer the fluid into 2 EP tubes, 150μl each.</p> | <p class="listing">e. Transfer the fluid into 2 EP tubes, 150μl each.</p> | ||
| - | <p class="listing">f. Add 300μl of solution II in each EP tube.</p> | + | <p class="listing">f. Add 300μl of solution II** in each EP tube.</p> |
| - | <p class="listing">g. Add 200μl of solution III in each EP tube and mix by inverting.</p> | + | <p class="listing">g. Add 200μl of solution III*** in each EP tube and mix by inverting.</p> |
<p class="listing">h. Centrifuge both EP tubes for 10 minutes at 14000rpm</p> | <p class="listing">h. Centrifuge both EP tubes for 10 minutes at 14000rpm</p> | ||
<p class="listing">i. Transfer 550μl of supernatant to column. </p> | <p class="listing">i. Transfer 550μl of supernatant to column. </p> | ||
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<p class="listing">q. Add 50μl of distilled water and centrifuge at 14000rpm for 1 minute.</p> | <p class="listing">q. Add 50μl of distilled water and centrifuge at 14000rpm for 1 minute.</p> | ||
<p class="listing">r. Keep filtered solution.</p> | <p class="listing">r. Keep filtered solution.</p> | ||
| - | <p class="listing_sub | + | <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 | + | <p class="listing_sub green">**solution Ⅱ 0.2N NaOH, 1% SDS</p> |
| - | <p class="listing_sub | + | <p class="listing_sub green">***solution Ⅲ 5M potassium acetate, glacial acetic acid, H₂O</p> |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 12:49, 17 June 2014
Miniprep
a. Transfer a single colony from the transformed E.colli to the LB medium and incubate in shaking incubator for 24 hours.
b. Centrifuge said E.colli for 15 minutes at 1500 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 14000rpm
i. Transfer 550μl of supernatant to column.
j. Centrifuge at 14000rpm for 1 minute.
k. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.
l. Centrifuge at 14000rpm for 1 minute.
m. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.
n. Centrifuge at 14000rpm for 1 minute.
o. Dispose of fluid in collection tube.
p. Centrifuge at 14000rpm for 1 minute.
q. Add 50μl of distilled water and centrifuge at 14000rpm 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
