Appendix
The protocols are reproduced directly from the manuals as is.
QIAprep Miniprep Handbook 05/2004
Protocol: QIAprep Spin Miniprep Kit 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. For purification of
low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using
other methods, refer to the recommendations on page 37.
Please read “Important Notes” on pages 19–21 before starting.
Note: All protocol steps should be carried out at room temperature.
Procedure
1. 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.
2. 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.
3. 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.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
A compact white pellet will form.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or
pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. (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 DH5£\™ do not require this additional wash step.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for
30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual
wash buffer.
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.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA,
add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep
spin column, let stand for 1 min, and centrifuge for 1 min.
QIAquick PCR Purification Kit Protocol
using a microcentrifuge
This protocol is designed to purify single- or double-stranded DNA fragments from PCR
and other enzymatic reactions (see page 8). For cleanup of other enzymatic reactions,
follow the protocol as described for PCR samples or use the new MinElute Reaction
Cleanup Kit. Fragments ranging from 100 bp to 10 kb are purified from primers,
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nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.
Notes: • Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• All centrifuge steps are at 13,000 rpm (~17,900 x g) in a conventional
tabletop microcentrifuge.
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary
to remove mineral oil or kerosene.
For example, add 500 µl of Buffer PB to 100 µl PCR sample (not including oil).
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
4. Discard flow-through. Place the QIAquick column back into the same tube.
Collection tubes are re-used to reduce plastic waste.
5. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
6. Discard flow-through and place the QIAquick column back in the same tube.
Centrifuge the column for an additional 1 min.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless
the flow-through is discarded before this additional centrifugation.
7. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of
the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for
increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick
membrane, let the column stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick
membrane for complete elution of bound DNA. The average eluate volume is 48 µl
from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within this
range, and store DNA at –20°C as DNA may degrade in the absence of a buffering
agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0),
but the EDTA may inhibit subsequent enzymatic reactions.
DNA from Plant
Tissue (Mini)
Protocol: Isolation of Total DNA from Plant Tissue Using
the DNeasy Plant Mini Kit
Important points before starting
If using the DNeasy Plant Mini Kit for the first time please read “Important Notes”
(page 12).
Buffer AP1 may develop a yellow color upon storage. This does not affect the
procedure.
All centrifugation steps are carried out at room temperature (15–25°C) in a microcentrifuge.
Things to do before starting
Buffers AP1 and AP3/E concentrate may form precipitates upon storage. If
necessary, warm to 65°C to redissolve (before adding ethanol to Buffer AP3/E).
Do not heat Buffer AP3/E after ethanol has been added.
Buffers AW and AP3/E are supplied as concentrates. Before using for the first time,
add the appropriate amount of ethanol (96–100%) as indicated on the bottle to
obtain a working solution.
Preheat a water bath or heating block to 65°C.
Manual disruption
Grind plant or fungal tissue under liquid nitrogen to a fine powder using a mortar and
pestle. Transfer the tissue powder and liquid nitrogen to an appropriately sized tube
and allow the liquid nitrogen to evaporate. Do not allow the sample to thaw. Proceed
immediately to the DNA preparation protocol.
Note: See “Disruption of plant tissue” (page 9).
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Disruption using the TissueLyser
Disruption of frozen tissue*
1. Place the sample material (≤100 mg wet weight) into a 2 ml Safe-Lock microtube,
together with a 5 mm stainless steel bead.
2. Freeze the tubes in liquid nitrogen for 30 s.
3. Place the tubes into the TissueLyser Adapter Set and fix into the clamps of the
TissueLyser. Immediately grind the samples for 1 min at 30 Hz.
4. Disassemble the Adaptor Set, remove the microtubes, and re-freeze the samples
in liquid nitrogen for 30 s.
DNA from Plant
Tissue (Mini)
5. Repeat step 3, reversing the position of the microtubes within the Adaptor Set.
To prevent variation in sample homogenization, the Adaptor Sets should be
removed from the TissueLyser and disassembled after the first disruption step. For
the second disruption step, the Adaptor Sets should be reassembled so that the
microtube order is reversed. Rotating the racks of collection microtubes in this way
ensures that all samples are thoroughly and equally disrupted.
Note: The majority of plant tissue is ground to a fine powder after two disruption
steps, however, for some materials one disruption step may be sufficient. Other
tissues, like seeds and roots, may need three disruption steps. Optimisation of the
disruption procedure may be required for some plant material.
Disruption of lyophilised tissue
Ensure that the material is dry otherwise grinding can lead to clumps.
1. Place the sample material (≤20 mg dry weight) into a 2 ml Safe-Lock microtube,
together with a 5 mm stainless steel bead.
2. Place the tubes into the TissueLyser Adapter Set and fix into the clamps of the
TissueLyser. Immediately grind the samples for 1 min at 30 Hz.
3. Disassemble the Adaptor Set, and reverse the positions of the microtubes. Grind
the samples for 1 min at 30 Hz.
To prevent variation in sample homogenization, the Adaptor Sets should be
removed from the TissueLyser and disassembled after the first disruption step. For
the second disruption step, the Adaptor Sets should be reassembled so that the
microtube order is reversed. Rotating the racks of collection microtubes in this way
ensures that all samples are thoroughly and equally disrupted.
DNA Preparation
1. Add 400 µl of Buffer AP1 and 4 µl of RNase A stock solution (100 mg/ml) to a
maximum of 100 mg of ground (wet weight) or 20 mg (dried) plant or fungal
tissue and vortex vigorously.
No tissue clumps should be visible. Vortex or pipet further to remove any clumps.
Clumped tissue will not lyse properly and will therefore result in a lower yield of
DNA. In the rare case where clumps cannot be removed by pipetting and vortexing,
a disposable micropestle may be used.
Note: Do not mix Buffer AP1 and RNase A before use.
2. Incubate the mixture for 10 min at 65°C. Mix 2–3 times during incubation by
inverting tube.
This step lyses the cells.
3. Add 130 µl of Buffer AP2 to the lysate, mix, and incubate for 5 min on ice.
This step precipitates detergent, proteins, and polysaccharides.
Optional: Centrifuge the lysate for 5 min at 20,000 x g (14,000 rpm).
Some plant materials can generate very viscous lysates and large amounts of
precipitates during this step resulting in shearing of the DNA in the next step (see
“Lysate filtration with QIAshredder”, page 13). In this case, optimal results are
obtained if the majority of these precipitates are removed by centrifugation for 5 min
at 20,000 x g (14,000 rpm). After centrifugation, apply supernatant to QIAshredder
Mini Spin Column and continue with step 4.
4. Apply the lysate to the QIAshredder Mini Spin Column (lilac) placed in a 2 ml
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collection tube and centrifuge for 2 min at 20,000 x g (14,000 rpm).
It may be necessary to cut the end off the pipet tip to apply the lysate to the
QIAshredder Mini Column. The QIAshredder Mini Column removes most
precipitates and cell debris, but a small amount will pass through and form a pellet
in the collection tube. Be careful not to disturb this pellet in step 5.
5. Transfer flow-through fraction from step 4 to a new tube (not supplied) without
disturbing the cell-debris pellet.
Typically 450 µl of lysate is recovered. For some plant species less lysate is recovered.
In this case determine volume for the next step.
6. Add 1.5 volumes of Buffer AP3/E to the cleared lysate and mix by pipetting.
Example: To 450 µl lysate add 675 µl Buffer AP3/E. Reduce the amount of Buffer
AP3/E accordingly if less lysate is recovered. A precipitate may form after the addition
of ethanol but this will not affect the DNeasy procedure.
Note: Ensure ethanol has been added to Buffer AP3/E (see “Things to do before
starting”).
Note: It is important to pipet Buffer AP3/E directly onto the cleared lysate and to
mix immediately.
7. Apply 650 µl of the mixture from step 6, including any precipitate which may have
formed, to the DNeasy Mini Spin Column sitting in a 2 ml collection tube (supplied).
Centrifuge for 1 min at ≥6000 x g (corresponds to ≥8000 rpm for most microcentrifuges)
and discard flow-through.*
Reuse the collection tube in step 8.
8. Repeat step 7 with remaining sample. Discard flow-through* and collection tube.
9. Place DNeasy Mini Spin Column in a new 2 ml collection tube (supplied), add 500 µl
Buffer AW to the DNeasy Mini Spin Column and centrifuge for 1 min at ≥6000 x g
(≥8000 rpm). Discard flow-through and reuse the collection tube in step 10.
Note: Ensure ethanol is added to Buffer AW.
10. Add 500 µl to the DNeasy Mini Spin Column and centrifuge for 2 min
at 20,000 x g (14,000 rpm) to dry the membrane.
It is important to dry the membrane of the DNeasy Mini Spin Column since residual
ethanol may interfere with subsequent reactions. This spin ensures that no residual
ethanol will be carried over during elution. Discard flow-through and collection
tube.
After washing with Buffer AW, the DNeasy Mini Spin Column membrane is usually
only slightly colored. In the rare case that the membrane remains significantly
colored after washing with Buffer AW, refer to “Darkly colored membrane” in the
Troubleshooting Guide on page 26.
Note: Following the spin, remove the DNeasy Mini Spin Column from the collection
tube carefully so the column does not come into contact with the flow-through, as
this will result in carryover of ethanol.
11. Transfer the DNeasy Mini Spin Column to a 1.5 ml or 2 ml microcentrifuge tube
(not supplied) and pipet 100 µl of Buffer AE directly onto the DNeasy membrane.
Incubate for 5 min at room temperature (15–25°C) and then centrifuge for 1 min
at ≥6000 x g (≥8000 rpm) to elute.
Elution with 50 µl (instead of 100 µl) increases the final DNA concentration in the
eluate significantly, but also reduces overall DNA yield. If larger amounts of DNA
(>20 µg) are loaded, eluting with 200 µl (instead of 100 µl) increases yield. See
“Elution” on page 13.
12. Repeat step 11 once.
A new microcentrifuge tube can be used for the second elution step to prevent
dilution of the first eluate. Alternatively, the microcentrifuge tube can be reused for
the second elution step to combine the eluates.
Note: More than 200 µl should not be eluted into a 1.5 ml microcentrifuge tube
because the DNeasy Mini Spin Column will come into contact with the eluate.
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GeneClean Turbo for PCR (QbioGene)
1. Transfer 100 µl oil-free PCR reaction to a 1.5 ml microcentrifuge tube.
2. Add 5 volumes GENECLEAN® Turbo for PCR Salt Solution and mix.
3. Transfer PCR/Salt Solution to a GENECLEAN® Turbo for PCR Cartridge.
4. Centrifuge for 5 seconds. Empty Catch Tube as needed.
5. Add 500 µl prepared GENECLEAN® Turbo for PCR Wash to the filter.
6. Centrifuge for 5 seconds. Empty Catch Tube as needed.
7. Empty Catch Tube. Centrifuge the GENECLEAN® Turbo for PCR Cartridge for an additional 4 minutes
to
remove residual Wash Solution.
8. Remove cap from Catch Tube, insert GENECLEAN® Turbo for PCR Cartridge containing bound DNA.
9. Add 30 µl GENECLEAN® Turbo for PCR Elution Solution directly onto GLASSMILK®-embedded
membrane
and incubate at room temperature for 5 minutes.
10. Centrifuge for 30 seconds to transfer eluted DNA to GENECLEAN® Turbo for PCR Catch Tube.
Discard
GENECLEAN® Turbo for PCR Cartridge and cap the Catch Tube.ENEC
Protocol
QIAquick Gel Extraction Kit Protocol
using a microcentrifuge
This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or
low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin
column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8).
For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer
QG and 1 volume of isopropanol to the reaction, mix, and proceed with step 6 of the
protocol. Alternatively, use the new MinElute Reaction Cleanup Kit.
Notes: • The yellow color of Buffer QG indicates a pH ≤7.5.
• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• Isopropanol (100%) and a heating block or water bath at 50°C are required.
• All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a
conventional table-top microcentrifuge.
• 3 M sodium acetate, pH 5.0, may be necessary.
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of
gel (100 mg ~ 100 µl).
For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose
gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick
column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help
dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is
yellow (similar to Buffer QG without dissolved agarose).
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5.
Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at
higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix.
For example, if the agarose gel slice is 100 mg, add 100 µl isopropanol. This step
increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments
between 500 bp and 4 kb, addition of isopropanol has no effect on yield.
Do not centrifuge the sample at this stage.
Protocol
6. Place a QIAquick spin column in a provided 2 ml collection tube.
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7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
The maximum volume of the column reservoir is 800 µl. For sample volumes of more
than 800 µl, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
Collection tubes are re-used to reduce plastic waste.
9. (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
This step will remove all traces of agarose. It is only required when the DNA will
subsequently be used for direct sequencing, in vitro transcription or microinjection.
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation
and direct sequencing, let the column stand 2–5 min after addition of Buffer PE,
before centrifuging.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min
at 13,000 rpm (~17,900 x g).
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless
the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the
QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased
DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane,
let the column stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick
membrane for complete elution of bound DNA. The average eluate volume is 48 µl
from 50 µl elution buffer volume, and 28 µl from 30 µl.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within
this range, and store DNA at –20°C as DNA may degrade in the absence of a
buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM
EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
CelLytic B II
Procedure
Trial Scale Purification
A small scale extraction trial should be performed to determine in which fraction your protein of interest
will be found. If this has already been determined, proceed to Large Scale Purification.
1. A 5 ml culture of the bacterial strain containing the recombinant protein should be grown in the
conditions for expression.
2. Use 1.5 ml of the bacterial culture with an OD600 of about 0.5-1.0 and centrifuge the cells at full speed
for 2 minutes.
3. Remove the medium and resuspend the cell pellet in 0.2 ml of CelLytic B II.
4. Vortex the mixture for 1-2 minutes to completely resuspend the cells and extract the soluble proteins
from the cells.
5. Centrifuge at full speed for 5 minutes to pellet the cell debris.
6. Carefully remove the soluble protein solution from the cell debris. This fraction contains about 90% of
the soluble protein. Additional extractions may be done, but this will result in a dilute protein solution.
7. Resuspend the cell pellet in 0.2 ml of CelLytic B II and vortex for 1-2 minutes to completely resuspend
the cell debris. Add 4 ml of the stock lysozyme solution to this extract (final concentration of lysozyme is
0.2 mg/ml).
8. Incubate at room temperature for 5-10 minutes to allow the lysozyme to fragment the cell wall.
9. Add 1 ml of the 20-fold diluted CelLytic B II to the tube and mix well.
10. Centrifuge at full speed for 5 minutes to pellet the cell debris again. Save the supernatant for analysis.
11. Resuspend the pellet in 1 ml of 20-fold diluted CelLytic B II and vortex for 1-2 minutes to completely
resuspend the remaining cell debris.
12. Centrifuge at full speed for 5 minutes to pellet the cell debris. Save the supernatant for analysis. Repeat
steps 11 and 12 two more times to completely remove the remaining soluble proteins and cell wall material
from the inclusion bodies.
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13. Resuspend the washed inclusion bodies in 0.4 ml deionized water or a buffer of choice.
14. Analyze all of the saved supernatants and the insoluble fraction by SDS-PAGE and/or immunoblot to
determine in which fraction your protein of interest is found. For SDS-PAGE, it is recommended that 2-8
ml of each of the samples be applied to the gel.
Large Scale Purification
This procedure is designed for 1 gram of wet cell paste. This amount of wet cell paste is roughly
equivalent to a 250 ml of bacterial culture with an OD600 of ~2.0. In order to extract the maximum amount
of soluble protein the cell mass to CelLytic B II ratio must be kept the same. This ratio is 5 ml of CelLytic
B II per gram of wet cell paste. Using less CelLytic B II will give a more concentrated solution, but a
smaller amount of total protein will be extracted. Using more CelLytic B II will not extract any more
protein; it will only serve to provide a more dilute protein solution. Therefore, it is best to adjust the volume
of CelLytic B II to the amount of the cell paste.
1. Collect the cells that express the protein of interest by centrifuging at 5,000 x g for 10 minutes.
2. Carefully remove the medium from the cell pellet. The cell pellet may be frozen or used fresh. A frozen
cell pellet will give a slightly higher yield of protein.
3. After adding the CelLytic B II (5 ml per g of cell paste), mix well to completely resuspend the cells.
Addition of Deoxyribonuclease I to a final concentration of 5 mg/ml will reduce the viscosity and yield a
solid pellet.
4. Incubate the extraction suspension with shaking at room temperature for 10-15 minutes to fully extract
the cells.
5. After the cells have been extracted, centrifuge the extract at 25,000 x g for 15 minutes to pellet the
insoluble material.
6. Carefully remove the supernatant that contains the soluble proteins. About 90 to 95% of the soluble
proteins will be found in this fraction. Another round of extraction will yield more soluble protein, but it
will be more dilute. If purifying an inclusion
body continue with this procedure.
Note: CelLytic B II will not make inclusion bodies soluble.
7. Resuspend the cell debris with another 5 ml of CelLytic B II. Mix the solution well to completely
resuspend the material. Add lysozyme to a final concentration of 0.4 mg/ml (200 ml of a 10 mg/ml stock
solution).
8. Incubate the mixture at room temperature for 5-10 minutes.
9. Add 30 ml of 20-fold diluted CelLytic B II to the extract and mix well.
10. Centrifuge the extract at 25,000 x g for 15 minutes to collect the inclusion bodies.
11. Carefully remove the supernatant from the inclusion bodies.
12. Resuspend the inclusion bodies in 40 ml of 20-fold diluted CelLytic B II.
13. Centrifuge the extract at 25,000 x g for 15 minutes to collect the inclusion bodies.
14. Repeat steps 12 and 13 two more times to further wash the inclusion bodies and then proceed on with
the protein purification.
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