Difference between revisions of "Team:NYU Shanghai/Protocols"
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<li>Column equilibration: add 500μl Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1min at 12,000 rpm. Discard the flow-through, and then place Spin Column CB1 back into the collection tube.</li> | <li>Column equilibration: add 500μl Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1min at 12,000 rpm. Discard the flow-through, and then place Spin Column CB1 back into the collection tube.</li> | ||
<li>Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.</li> | <li>Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.</li> | ||
− | <li>Transfer the mixture to the Spin Column CB1, incubate at room temperature for 2min. Centrifuge for 30-60s at 12,000rpm. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube. | + | <li>Transfer the mixture to the Spin Column CB1, incubate at room temperature for 2min. Centrifuge for 30-60s at 12,000rpm. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube. |
− | <br>The maximum loading volume of the column is 800μl. For sample volumes greater than 800 μl simply load again. | + | <br>The maximum loading volume of the column is 800μl. For sample volumes greater than 800 μl simply load again.</li> |
− | <li>Add 600 μl Buffer PW (ensure that ethanol has been added) to the Spin Column CB1 and centrifuge for 30-60s at 12,000 rpm. Discard the flow-through, and place Spin Column CB1 back in the same collection tube. | + | <li>Add 600 μl Buffer PW (ensure that ethanol has been added) to the Spin Column CB1 and centrifuge for 30-60s at 12,000 rpm. Discard the flow-through, and place Spin Column CB1 back in the same collection tube. |
− | <br>Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5min after adding Buffer PW, and then centrifuge. | + | <br>Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5min after adding Buffer PW, and then centrifuge.</li> |
<li>Repeat step 4.</li> | <li>Repeat step 4.</li> | ||
<li>Centrifuge at 12,000 rpm for 2min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.</li> | <li>Centrifuge at 12,000 rpm for 2min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.</li> |
Revision as of 00:54, 18 September 2015