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Team:KULeuven/Protocols
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* [[Team:KULeuven/Protocols/Miniprep|Miniprep (Qiagen)]] | * [[Team:KULeuven/Protocols/Miniprep|Miniprep (Qiagen)]] | ||
* ... | * ... | ||
| + | |||
| + | == Plasmid DNA purification == | ||
| + | |||
| + | === Materials === | ||
| + | * LB broth with appropriate antibiotic | ||
| + | * 15 ml tube | ||
| + | * incubation oven at 37°C | ||
| + | * microcentifuge tubes | ||
| + | * table-top microcentrifuge | ||
| + | * buffer P1 (Qiagen kit) | ||
| + | * buffer P2 (Qiagen kit) | ||
| + | * buffer N3 (Qiagen kit) | ||
| + | * QIAprep spin columns | ||
| + | * transparant tape | ||
| + | * buffer PE (Qiagen kit) | ||
| + | * buffer EB (Qiagen kit) | ||
| + | * nanodrop | ||
| + | |||
| + | === Procedure === | ||
| + | |||
| + | # Inoculate a single colony into 5 ml of LB with the appropriate antibiotic and incubate at 37°C for 12-16 hours (liquid culture). | ||
| + | # Put 1.5 ml of this liquid culture in a microcentrifuge tube and centrifuge at 8500 rpm for 3 minutes at room temperature. | ||
| + | # Remove all traces of supernatant by inverting the tube. | ||
| + | # Resuspend the pelleted cells in 250 $\mu$l Buffer P1 and vortex until no cell clumps remain. | ||
| + | # Add 250 ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times. !! Do not vortex and do not allow the reaction to proceed for more than 5 minutes ! | ||
| + | # Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The solution will become cloudy. | ||
| + | # Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will form. | ||
| + | # Apply the supernatant of step 4 to a QIAprep spin column by decanting. When you label the columns, cover this label with tape (the ink tends to dissolve). | ||
| + | # Centrifuge for 1 minute at 13000 rpm. Discard the flow-through. If you made several tubes of one sample, you can repeat the steps 8 and 9 in the same QIAprep spin column. This way, the concentration of plasmid DNA will be higher. | ||
| + | # Wash the QIAprep spin column with 0.75 ml Buffer PE. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through !! | ||
| + | # Centrifuge for an additional minute to remove all residual wash buffer. | ||
| + | # Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 ul Buffer EB to the centre of the QIAprep spin column, let stand for 1 minute and centrifuge for 1 minute at 13000 rpm. | ||
| + | # Measure the concentration of plasmid DNA with the nanodrop (ng/ul). Use Buffer EB as blank. | ||
Revision as of 20:36, 8 October 2008
dock/undock dropdown
Contents |
Protocols Wet Lab
Plasmid DNA purification
Materials
- LB broth with appropriate antibiotic
- 15 ml tube
- incubation oven at 37°C
- microcentifuge tubes
- table-top microcentrifuge
- buffer P1 (Qiagen kit)
- buffer P2 (Qiagen kit)
- buffer N3 (Qiagen kit)
- QIAprep spin columns
- transparant tape
- buffer PE (Qiagen kit)
- buffer EB (Qiagen kit)
- nanodrop
Procedure
- Inoculate a single colony into 5 ml of LB with the appropriate antibiotic and incubate at 37°C for 12-16 hours (liquid culture).
- Put 1.5 ml of this liquid culture in a microcentrifuge tube and centrifuge at 8500 rpm for 3 minutes at room temperature.
- Remove all traces of supernatant by inverting the tube.
- Resuspend the pelleted cells in 250 $\mu$l Buffer P1 and vortex until no cell clumps remain.
- Add 250 ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times. !! Do not vortex and do not allow the reaction to proceed for more than 5 minutes !
- Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The solution will become cloudy.
- Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will form.
- Apply the supernatant of step 4 to a QIAprep spin column by decanting. When you label the columns, cover this label with tape (the ink tends to dissolve).
- Centrifuge for 1 minute at 13000 rpm. Discard the flow-through. If you made several tubes of one sample, you can repeat the steps 8 and 9 in the same QIAprep spin column. This way, the concentration of plasmid DNA will be higher.
- Wash the QIAprep spin column with 0.75 ml Buffer PE. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through !!
- Centrifuge for an additional minute to remove all residual wash buffer.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 ul Buffer EB to the centre of the QIAprep spin column, let stand for 1 minute and centrifuge for 1 minute at 13000 rpm.
- Measure the concentration of plasmid DNA with the nanodrop (ng/ul). Use Buffer EB as blank.
