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Team:Macquarie Australia/Protocols/Plasmid Prep
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| - | + | <center><b>QIAprep Spin Miniprep Kit Protocol</b></center><br><br> | |
| - | + | 1) Pellet 1-5 mL bacterial culture by centrifugation at >8000 rpm for 3 minutes at room temperature.<br><br> | |
| - | + | 2) Resuspend Pelleted bacterial cells in 250 uL Buffer P1.<br><br> | |
| - | + | 3) Add 250 uL Buffer P2 and invert tube 4-6 times.<br><br> | |
| - | + | 4) Add 350 uL Buffer N3 and invert immediately 4-6 times and centrifuge for 10 minutes at 13,000 rpm.<br><br> | |
| - | + | 5) Apply the supernatant to a QIAprep spin column and centrifuge for 30 seconds. Discard the flow-through.<br><br> | |
| - | 9) Place the QIAprep spin column into a clean Eppendorf. Elute DNA by adding 50 uL Buffer EB directly to the filter. Tap the column and stand for 1 minute and then centrifuge for 1 minute. | + | 6) Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.<br><br> |
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| + | 7) Wash the QIAprep spin column with 0.75 mL Buffer PE and centrifuge for 30 seconds. Discard the flow-through.<br><br> | ||
| + | |||
| + | 8) Centrifuge for 1 minute to remove residual wash buffer.<br><br> | ||
| + | |||
| + | 9) Place the QIAprep spin column into a clean Eppendorf. Elute DNA by adding 50 uL Buffer EB directly to the filter. Tap the column and stand for 1 minute and then centrifuge for 1 minute.<br><br> | ||
Revision as of 04:50, 29 August 2013
Plasmid Prep
1) Pellet 1-5 mL bacterial culture by centrifugation at >8000 rpm for 3 minutes at room temperature.
2) Resuspend Pelleted bacterial cells in 250 uL Buffer P1.
3) Add 250 uL Buffer P2 and invert tube 4-6 times.
4) Add 350 uL Buffer N3 and invert immediately 4-6 times and centrifuge for 10 minutes at 13,000 rpm.
5) Apply the supernatant to a QIAprep spin column and centrifuge for 30 seconds. Discard the flow-through.
6) Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.
7) Wash the QIAprep spin column with 0.75 mL Buffer PE and centrifuge for 30 seconds. Discard the flow-through.
8) Centrifuge for 1 minute to remove residual wash buffer.
9) Place the QIAprep spin column into a clean Eppendorf. Elute DNA by adding 50 uL Buffer EB directly to the filter. Tap the column and stand for 1 minute and then centrifuge for 1 minute.
