"
Team:Macquarie Australia/Protocols/Plasmid Prep
From 2013.igem.org
| Line 4: | Line 4: | ||
<center> | <center> | ||
<h1>Plasmid Prep</h1> | <h1>Plasmid Prep</h1> | ||
| + | <b>QIAprep Spin Miniprep Kit Protocol</b> | ||
</center> | </center> | ||
| - | + | [[File:PlasmidPrep.jpg|500px|thumb|left|Plasmid Prep diagram from http://solgent.com/english/QC/831]] | |
| + | <html> | ||
| - | |||
| - | 1) Pellet 1-5 mL bacterial culture by centrifugation at >8000 rpm for 3 minutes at room temperature.<br><br> | + | <b>1)</b> 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> | + | <b>2)</b> 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> | + | <b>3)</b> 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> | + | <b>4)</b> 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> | + | <b>5)</b> Apply the supernatant to a QIAprep spin column and centrifuge for 30 seconds. Discard the flow-through.<br><br> |
| - | 6) Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.<br><br> | + | <b>6)</b> Wash the QIAprep spin column with 0.5 mL Buffer PB and centrifuge for 30 seconds. Discard the flow-through.<br><br> |
| - | 7) Wash the QIAprep spin column with 0.75 mL Buffer PE and centrifuge for 30 seconds. Discard the flow-through.<br><br> | + | <b>7)</b> 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> | + | <b>8)</b> 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> | + | <b>9)</b> 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 06:02, 13 September 2013
Plasmid Prep
QIAprep Spin Miniprep Kit Protocol
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.
