Nov 15, 2015
Qiagen QIAprep Spin Miniprep Kit (27104 or 27106) with a microcentrifuge
Forked from Modified Qiagen QIAprep Spin Miniprep
- QIAgen1
- 1Stony Brook University
External link: https://www.qiagen.com/us/resources/resourcedetail?id=22df6325-9579-4aa0-819c-788f73d81a09&lang=en
Protocol Citation: QIAgen 2015. Qiagen QIAprep Spin Miniprep Kit (27104 or 27106) with a microcentrifuge. protocols.io https://dx.doi.org/10.17504/protocols.io.d7f9jm
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 15, 2015
Last Modified: March 21, 2018
Protocol Integer ID: 1991
Abstract
This is the protocol for QIAgen's QIAprep Spin Miniprep Kit (catalog numbers 27104 or 27106) with their newer version 2.0 spin columns. For more information about the kit, see https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiaprep-spin-miniprep-kit?cmpid=QVenSPP1404quartzySC#orderinginformation.
For purification of up to 20 μg molecular biology grade plasmid DNA
Ready-to-use plasmid DNA in minutes
Reproducible yields of molecular biology grade plasmid DNA
Single protocol for high- and low-copy vectors
Even higher yields with the High-Yield Supplementary Protocol
Improved QIAprep 2.0 Spin Column
GelPilot loading dye for convenient sample analysis
The QIAprep Spin Miniprep Kit is designed for isolation of up to 20 μg high-purity plasmid or cosmid DNA for use in routine molecular biology applications, including fluorescent and radioactive sequencing and cloning. Even higher yields (up to 30 μg) can be achieved using the High-Yield Supplementary Protocol.
Attachments
Guidelines
- Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from CO2 in the air.
- All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional, table-top microcentrifuge.
- Ensure that the elution buffer is dispensed directly onto the center of the QIAprep membrane for optimal elution of DNA. Average eluate volume is 48 μl from an elution buffer volume of 50 μl (QIAprep spin procedures), and 60 μl from an elution buffer volume of 100 μl (QIAprep multiwell procedures).
- For increased DNA yield, use a higher elution-buffer volume.
- For increased DNA concentration, use a lower elution-buffer volume
- If water is used for elution, make sure that its pH is between 7.0 and 8.5. Elution efficiency is dependent on pH and the maximum elution efficiency is achieved within this range. A pH <7.0 can decrease yield. Note: Store DNA at –20°C when eluted with water, as DNA may degrade in the absence of a buffering agent. DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
Safety warnings
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. Buffers P2, N3, and PB contain irritants. Wear gloves when handling these buffers. For more information, please consult the appropriate safety data sheets (SDSs).
CAUTION:DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
Buffers N3 and PB contain guanidine hydrochloride, which can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
Before start
Growth of bacterial cultures in tubes or flasks
Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking. Growth for more than 16 h is not recommended since cells begin to lyse and plasmid yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture.
- Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A (centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 μg/ml. Mix and store at 2–8°C.
- Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
- Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to 37°C. Do not shake Buffer P2 vigorously.
- Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 μl LyseBlue into 10 ml Buffer P1). LyseBlue provides visual identification of optimum buffer mixing, thereby preventing the common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris.
Lysis
Lysis
Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a micro-centrifuge tube.
250 µL
Note
Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
250 µL
Note
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.
If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
350 µL
Note
To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless sus- pension indicates that the SDS has been effectively precipitated.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
00:10:00
Binding
Binding
Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
Note
Roughly about 850 uL.
Centrifuge for 30–60 s. Discard the flow-through.
00:01:00
Wash
Wash
Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
500 µL
00:01:00
Note
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 DH5®α do not require this additional wash step.
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
750 µL
00:01:00
Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer
00:01:00
Note
Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
Elution
Elution
Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
00:01:00
Centrifuge for 1 min.
00:01:00
Let stand 1 minute
00:01:00
Lysis
Lysis
Centrifuge 1-5mL bacterial overnight culture at >6800g for 3 minutes at room temperature
5 mL