Team:UIUC-Illinois/Notebook/Protocols/Miniprep
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- | <center><h1> | + | <center><h1>Miniprep</h1></center><br/> |
+ | A miniprep is done to purify plasmids from a strain <br/><br/> | ||
+ | What you need:<br/> | ||
+ | - An overnight culture of your plasmid-containing cells<br/> | ||
+ | - Autoclaved 1.5mL centrifuge tubes<br/> | ||
+ | - Resuspension solution containing RNase A<br/> | ||
+ | - Lysis solution<br/> | ||
+ | - Neutralization solution<br/> | ||
+ | - Large Zymo-spin columns with collection tubes<br/> | ||
+ | - DNA wash buffer<br/> | ||
+ | - Autoclaved MilliQ water<br/><br/> | ||
+ | Procedure:<br/><br/> | ||
+ | 1. To a 1.5 mL centrifuge tube, add 1.5 mL of overnight culture<br/> | ||
+ | 2. Centrifuge at full speed for 2 minutes and discard supernatant<br/> | ||
+ | 3. Resuspend the cells by pipetting in 250 uL resuspension solution<br/> | ||
+ | 4. Add 250 uL lysis solution and mix by inversion (about 30 inversions)<br/> | ||
+ | 5. Add 350 uL neutralization solution and mix by inversion<br/> | ||
+ | 6. Centrifuge the mixture at full speed for 5 minutes<br/> | ||
+ | 7. Transfer the supernatant to a Zymo-spin column with collection tube<br/> | ||
+ | 8. Centrifuge for 1 minute and discard the flow-through in collection tube<br/> | ||
+ | 9. Add 500 uL wash buffer to column<br/> | ||
+ | 10. Centrifuge for 1 minute and discard the supernatant<br/> | ||
+ | 11. Repeat steps 9-10<br/> | ||
+ | 12. Spin the empty column for an additional minute<br/> | ||
+ | 13. Place the column in a new 1.5 mL centrifuge tube<br/> | ||
+ | 14. Add 50 uL autoclaved water directly to the column<br/> | ||
+ | 15. Let the column incubate at room temperature for 2 minutes<br/> | ||
+ | 16. Spin the column in the centrifuge for 2 minutes<br/> | ||
+ | 17. Store the liquid at -20˚C until use<br/><br/> | ||
+ | |||
</div> | </div> | ||
Revision as of 15:08, 4 June 2012