Team:Aberdeen Scotland/Mini Preps

From 2009.igem.org

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==Protocol==
==Protocol==
1. Culture E.coli overnight in 5ml LB medium in a shaking incubator at 37C
1. Culture E.coli overnight in 5ml LB medium in a shaking incubator at 37C
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2. Centrifuge culture at 4000 rpm for 10 minutes.
2. Centrifuge culture at 4000 rpm for 10 minutes.
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3. Resuspend pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube.
3. Resuspend pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube.
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4. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times
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4. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
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5. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting tube 4-6 times.
5. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting tube 4-6 times.
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6. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
6. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
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7. Apply supernatant to QIAprep spin column by decanting/pipetting
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7. Apply supernatant to QIAprep spin column by decanting/pipetting.
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8. Centrifuge for 60s at 13,000 rpm and discard flow-through.
8. Centrifuge for 60s at 13,000 rpm and discard flow-through.
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9. Wash column by adding 0.5ml Buffer PB, centrifuge for 60s and discard flow-through.
9. Wash column by adding 0.5ml Buffer PB, centrifuge for 60s and discard flow-through.
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10. Wash column by adding 0.75ml Buffer PE, centrifuge for 60s and discard flow-through.
10. Wash column by adding 0.75ml Buffer PE, centrifuge for 60s and discard flow-through.
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<br>
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11. Centrifuge for an additional minute and discard flow-through.
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<br>
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12. To elute DNA, place QIAprep column in clean 1.5ml microcentrifuge tube.  Add 50µl Buffer EB (or water) to center of each QIAprep spin column, let stand for 2 min and centrifuge for 1 min.
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Revision as of 14:40, 11 August 2009

University of Aberdeen iGEM 2009

Mini Preps

Our mini preps were prepared using QIAprep Spin Miniprep Kits from Qiagen. The protocol is from the handbook, using a Microcentrifuge.

Protocol

1. Culture E.coli overnight in 5ml LB medium in a shaking incubator at 37C
2. Centrifuge culture at 4000 rpm for 10 minutes.
3. Resuspend pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube.
4. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
5. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting tube 4-6 times.
6. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
7. Apply supernatant to QIAprep spin column by decanting/pipetting.
8. Centrifuge for 60s at 13,000 rpm and discard flow-through.
9. Wash column by adding 0.5ml Buffer PB, centrifuge for 60s and discard flow-through.
10. Wash column by adding 0.75ml Buffer PE, centrifuge for 60s and discard flow-through.
11. Centrifuge for an additional minute and discard flow-through.
12. To elute DNA, place QIAprep column in clean 1.5ml microcentrifuge tube. Add 50µl Buffer EB (or water) to center of each QIAprep spin column, let stand for 2 min and centrifuge for 1 min.

  Gel Preperation Purification  

 

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