Duke University/27 June 2009
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(New page: Harvested bacteria <br> Did mini-prep) |
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- | + | [[Team:Duke | Return]] | |
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+ | ==June 27, 2009== | ||
+ | Harvest bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for 2 min at RT. Decant supernatant and remove remaining medium. | ||
+ | |||
+ | Purification/Mini prep | ||
+ | # Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or pipet up and down until no cell clumps remain. | ||
+ | # Add 250 Lysis Solution and mix thoroughly by inverting tube 4-6 times until solution becomes viscous and slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA. | ||
+ | # Add 350 ul Neutralization Solution and mix thoroughly by inverting tube 4-6 times. | ||
+ | # Centrifuge for 5 min to pellet cell debris and chromosomal DNA. | ||
+ | # Transfer supernatant to spin column by decanting or pipetting. Avoid disturbing or transferring white precipitate. | ||
+ | # Centrifuge for 1 min. Discard flow-through and replace column in collection tube. | ||
+ | # Add 500 ul Wash Solution to spin column. Centrifuge for 30-60s and discard flow-through. | ||
+ | # Repeat step 7. | ||
+ | # Centrifuge for 1 min to remove residual Wash Solution. | ||
+ | # Transfer spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to center of spin column membrane. Do not touch pipette tip to membrane. Incubate 2 min at RT and centrifuge for 2 min. | ||
+ | Note: An additional elution step with Elution buffer or water will recover residual DNA from the membrane and increase overall yield by 10-20%. | ||
+ | # Discard column and store purified plasmid DNA at -20°C. |
Latest revision as of 17:30, 21 October 2009
June 27, 2009
Harvest bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for 2 min at RT. Decant supernatant and remove remaining medium.
Purification/Mini prep
- Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or pipet up and down until no cell clumps remain.
- Add 250 Lysis Solution and mix thoroughly by inverting tube 4-6 times until solution becomes viscous and slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
- Add 350 ul Neutralization Solution and mix thoroughly by inverting tube 4-6 times.
- Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
- Transfer supernatant to spin column by decanting or pipetting. Avoid disturbing or transferring white precipitate.
- Centrifuge for 1 min. Discard flow-through and replace column in collection tube.
- Add 500 ul Wash Solution to spin column. Centrifuge for 30-60s and discard flow-through.
- Repeat step 7.
- Centrifuge for 1 min to remove residual Wash Solution.
- Transfer spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to center of spin column membrane. Do not touch pipette tip to membrane. Incubate 2 min at RT and centrifuge for 2 min.
Note: An additional elution step with Elution buffer or water will recover residual DNA from the membrane and increase overall yield by 10-20%.
- Discard column and store purified plasmid DNA at -20°C.