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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. | ||
| - | 4. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times | + | <br> |
| + | 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. | ||
| - | 7. Apply supernatant to QIAprep spin column by decanting/pipetting | + | <br> |
| + | 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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| + | 11. Centrifuge for an additional minute and discard flow-through. | ||
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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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<html> | <html> | ||
<table class="nav"> | <table class="nav"> | ||
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| - | <a href="https://2009.igem.org/Team:Aberdeen_Scotland/ | + | <a href="https://2009.igem.org/Team:Aberdeen_Scotland/Digestion"><img src="https://static.igem.org/mediawiki/2009/e/ed/Aberdeen_Left_arrow.png"> Digestion</a> |
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<td align="right"> | <td align="right"> | ||
Latest revision as of 14:14, 17 August 2009
University of Aberdeen - Pico Plumber
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.
 Digestion
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Purification 
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