Team:Newcastle/Minipreps
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{{Team:Newcastle/mainbanner}} | {{Team:Newcastle/mainbanner}} | ||
- | =Minipreps= | + | =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== | ==Procedures== | ||
- | + | # Overnight culture should have been done the day before. Refer to [[Team:Newcastle/Growing an overnight cultures| Growing an overnight culture]]. | |
# Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. | # 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 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times. |
Revision as of 15:03, 29 July 2010
|
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 min at 13,000 rpm in a table-top microcentrifuge.
- Apply the supernatant to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30-60s. Discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s.
- Discard the flow-through, and centrifuge for an additional 1 min 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 min, and centrifuge for 1 min.