Team:Newcastle/Minipreps
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{{Team:Newcastle/mainbanner}} | {{Team:Newcastle/mainbanner}} | ||
- | + | =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 [[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. |
- | # | + | # 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. | |
+ | |||
+ | |||
+ | '''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' | ||
+ | |||
+ | {{Team:Newcastle/footer}} |
Latest revision as of 12:54, 2 August 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 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.
Go back to our Protocol List