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Team:RHIT/Protocols/Miniprep
From 2014.igem.org
QIAprep Spin Miniprep Kit Protocol
using a microcentrifuge
This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium.
Note: All protocol steps should be carried out at room temperature
Procedure
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
- Add 250 µl Buffer P2 and gently invert the tube 4-6 times to mix.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
- Add 350 µl Buffer N3 and invert the tube immediately but gently 4-6 times.
To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
A compact white pellet will form.
- Apply the supernatants from step 4 to the QIAprep Spin Column by decanting or pipetting.
- Centrifuge for 30–60 s. Discard the flow-through.
- (Optional): Wash the QIAprep Spin Column by adding 0.5 ml Buffer PB and
centrifuging for 30–60 s. Discard the flow-through.
This step is necessary to remove trace nuclease activity when using endA+strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step.
- Wash QIAprep Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
