Team:Newcastle/Minipreps
From 2010.igem.org
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# Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times. | # Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times. | ||
# Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. | # Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. | ||
- | # Centrifuge for 10 | + | # Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge. |
# Apply the supernatant to the QIAprep spin column by decanting or pipetting. | # Apply the supernatant to the QIAprep spin column by decanting or pipetting. | ||
- | # Centrifuge for 30- | + | # Centrifuge for 30-60 seconds. Discard the flow-through. |
- | # Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30- | + | # Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through. |
- | # Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30- | + | # Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds. |
- | # Discard the flow-through, and centrifuge for an additional 1 | + | # Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer. |
- | # To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 | + | # To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute. |
{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} |
Revision as of 15:04, 29 July 2010
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Minipreps using the Qiagen kit
Materials required
- Eppendorf tubes
- Pipettes
- Appropriate overnight cultures
- Buffer P1 (In the fridge)
- Buffer P2
- Buffer N3
- Buffer PB
- Buffer EB
- QIAprep spin column
Procedures
- Overnight culture should have been done the day before. Refer to Growing an overnight culture.
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
- Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
- Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
- Apply the supernatant to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30-60 seconds. Discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds.
- Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer.
- To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.