Team:Newcastle/Minipreps
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RachelBoyd (Talk | contribs) (New page: ==Minipreps== ===Gel extraction=== #Excise the DNA fragment from the agarose gel with a clean, sharp scalpel #Weigh the gel slice in the colourless tube. Add 3 volumes Buffer QG to 1 vol...) |
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- | + | {{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
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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.
Go back to our Protocol List