Mini and Midi prep

From 2007.igem.org

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inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centrifuge
inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centrifuge
with rotor and tubes or bottles for harvesting cells)
with rotor and tubes or bottles for harvesting cells)
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■ QIArack or equivalent holder (see “Setup of QIAGEN-tips”, page 13)
■ QIArack or equivalent holder (see “Setup of QIAGEN-tips”, page 13)
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■ Ice
■ Ice
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■ Isopropanol
■ Isopropanol
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■ 70% ethanol
■ 70% ethanol
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■ Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)
■ Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)
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For QIAGEN Plasmid Mini Kit protocol:
 
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■ Microcentrifuge
 
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■ 1.5 ml or 2 ml microcentrifuge tubes
 
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For QIAGEN Plasmid Midi, Maxi, Mega, and Giga Kit protocols:
 
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■ Centrifugation tubes or vessels with suitable capacity for the volumes specified in the
 
-
appropriate protocol.
 
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■ Refrigerated centrifuge capable of ≥20,000 x g with rotor for the appropriate
 
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centrifuge tubes or bottles
 
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[[Protocol: Plasmid or Cosmid DNA Purification Using
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''For QIAGEN Plasmid Mini Kit protocol:''
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QIAGEN Plasmid Mini Kit]]
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■ Microcentrifuge
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■ 1.5 ml or 2 ml microcentrifuge tubes
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''For QIAGEN Plasmid Midi, Maxi, Mega, and Giga Kit protocols:''
 +
■ Centrifugation tubes or vessels with suitable capacity for the volumes specified in the
 +
appropriate protocol.
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[[Protocol: Plasmid or Cosmid DNA Purification Using
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■ Refrigerated centrifuge capable of ≥20,000 x g with rotor for the appropriate
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QIAGEN Plasmid Midi and Maxi Kits]]
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centrifuge tubes or bottles
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''Procedure''
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[[Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit]]
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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter
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[[Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits]]
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culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate
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for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).
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Use a tube or flask with a volume of at least 4 times the volume of the culture.
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2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy
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plasmids, inoculate ▲ 25 ml or ● 100 ml medium with ▲ 25–50 μl or
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● 100–200 μl of starter culture. For low-copy plasmids, inoculate ▲ 100 ml or
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● 500 ml medium with ▲ 100–200 μl or ● 250–500 μl of starter culture. Grow at
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37°C for 12–16 h with vigorous shaking (approx. 300 rpm).
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Use a flask or vessel with a volume of at least 4 times the volume of the culture. The
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culture should reach a cell density of approximately 3–4 x 109 cells per milliliter,
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which typically corresponds to a pellet wet weight of approximately 3 g/liter
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medium.
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3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
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ƒ If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
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4. Resuspend the bacterial pellet in ▲ 4 ml or ● 10 ml Buffer P1.
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For efficient lysis it is important to use a vessel that is large enough to allow complete
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mixing of the lysis buffers. Ensure that RNase A has been added to Buffer P1.
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If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle
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before use to ensure LyseBlue particles are completely resuspended. The bacteria
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should be resuspended completely by vortexing or pipetting up and down until no
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cell clumps remain.
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5. Add ▲ 4 ml or ● 10 ml Buffer P2, mix thoroughly by vigorously inverting the sealed
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tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min.
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Do not vortex, as this will result in shearing of genomic DNA. The lysate should
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appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After
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use, the bottle containing Buffer P2 should be closed immediately to avoid
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acidification from CO2 in the air.
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If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after
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addition of Buffer P2. Mixing should result in a homogeneously colored suspension.
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If the suspension contains localized colorless regions or if brownish cell clumps are
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still visible, continue mixing the solution until a homogeneously colored suspension
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is achieved.
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6. Add ▲ 4 ml or ● 10 ml of chilled Buffer P3, mix immediately and thoroughly by
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vigorously inverting 4–6 times, and incubate on ice for ▲ 15 min or ● 20 min.
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Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After
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addition of Buffer P3, a fluffy white material forms and the lysate becomes less
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viscous. The precipitated material contains genomic DNA, proteins, cell debris, and
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KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulfate
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precipitation. If the mixture still appears viscous, more mixing is required to
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completely neutralize the solution.
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If LyseBlue reagent has been used, the suspension should be mixed until all trace of
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blue has gone and the suspension is colorless. A homogeneous colorless suspension
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indicates that the SDS has been effectively precipitated.
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7. Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid
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DNA promptly.
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Before loading the centrifuge, the sample should be mixed again. Centrifugation
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should be performed in non-glass tubes (e.g., polypropylene). After centrifugation
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the supernatant should be clear.
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Note: Instead of centrifugation steps 7 and 8, the lysate can be efficiently cleared by
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filtration using a QIAfilter Kits or Cartridges (see www.qiagen.com/products/
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plasmid/LargeScaleKits).
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8. Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove
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supernatant containing plasmid DNA promptly.
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This second centrifugation step should be carried out to avoid applying suspended
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or particulate material to the QIAGEN-tip. Suspended material (causing the sample
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to appear turbid) can clog the QIAGEN-tip and reduce or eliminate gravity flow.
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☞ Remove a ▲ 240 μl or ● 120 μl sample from the cleared lysate supernatant and
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save for an analytical gel (sample 1) in order to determine whether growth and
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lysis conditions were optimal.
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9. Equilibrate a ▲ QIAGEN-tip 100 or ● QIAGEN-tip 500 by applying ▲ 4 ml or
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● 10 ml Buffer QBT, and allow the column to empty by gravity flow.
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Flow of buffer will begin automatically by reduction in surface tension due to the
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presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain
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completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop
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when the meniscus reaches the upper frit in the column.
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10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin
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by gravity flow.
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The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long
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and becomes cloudy due to further precipitation of protein, it must be centrifuged
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again or filtered before loading to prevent clogging of the QIAGEN-tip.
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☞ Remove a ▲ 240 μl or ● 120 μl sample from the flow-through and save for an
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analytical gel (sample 2) in order to determine the efficiency of DNA binding to
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the QIAGEN Resin.
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11. Wash the QIAGEN-tip with ▲ 2 x 10 ml or ● 2 x 30 ml Buffer QC.
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Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first wash is
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sufficient to remove all contaminants in the majority of plasmid DNA preparations.
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The second wash is especially necessary when large culture volumes or bacterial
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strains producing large amounts of carbohydrates are used.
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☞ Remove a ▲ 400 μl or ● 240 μl sample from the combined wash fractions and
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save for an analytical gel (sample 3).
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12. Elute DNA with ▲ 5 ml or ● 15 ml Buffer QF.
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Collect the eluate in a 15 ml or 50 ml tube (not supplied). Use of polycarbonate
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centrifuge tubes is not recommended as polycarbonate is not resistant to the alcohol
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used in subsequent steps.
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Note: For constructs larger than 45–50 kb, prewarming the elution buffer to 65°C
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may help to increase yield.
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☞ Remove a ▲ 100 μl or ● 60 μl sample of the eluate and save for an analytical
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gel (sample 4).
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ƒ If you wish to stop the protocol and continue later, store the eluate at 4°C. Storage
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periods longer than overnight are not recommended.
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13. Precipitate DNA by adding ▲ 3.5 ml or ● 10.5 ml (0.7 volumes) room-temperature
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isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for
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30 min at 4°C. Carefully decant the supernatant.
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All solutions should be at room temperature in order to minimize salt precipitation,
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although centrifugation is carried out at 4°C to prevent overheating of the sample.
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Alternatively, disposable conical bottom centrifuge tubes can be used for
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centrifugation at 5000 x g for 60 min at 4°C. Isopropanol pellets have a glassy
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appearance and may be more difficult to see than the fluffy, salt-containing pellets
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that result from ethanol precipitation. Marking the outside of the tube before
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centrifugation allows the pellet to be more easily located. Isopropanol pellets are also
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more loosely attached to the side of the tube, and care should be taken when
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removing the supernatant.
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14. Wash DNA pellet with ▲ 2 ml or ● 5 ml of room-temperature 70% ethanol, and
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centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without
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disturbing the pellet.
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Alternatively, disposable conical-bottom centrifuge tubes can be used for
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centrifugation at 5000 x g for 60 min at 4°C. The 70% ethanol removes precipitated
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salt and replaces isopropanol with the more volatile ethanol, making the DNA easier
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to redissolve.
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15. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer
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(e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
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Redissolve the DNA pellet by rinsing the walls to recover all the DNA, especially if
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glass tubes have been used. Pipetting the DNA up and down to promote
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resuspension may cause shearing and should be avoided. Overdrying the pellet will
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make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline
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conditions; it does not easily dissolve in acidic buffers.
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Determination of yield
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To determine the yield, DNA concentration should be determined by both UV
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spectrophotometry at 260 nm and quantitative analysis on an agarose gel. For reliable
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spectrophotometric DNA quantification, A260 readings should lie between 0.1 and 1.0.
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Agarose gel analysis
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We recommend removing and saving aliquots during the purification procedure
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(samples 1–4). If the plasmid DNA is of low yield or quality, the samples can be
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analyzed by agarose gel electrophoresis to determine at what stage of the purification
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procedure the problem occurred
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Latest revision as of 15:45, 20 September 2007

Equipment and Reagents to Be Supplied by User

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.

For all protocols:

■ Standard microbiological equipment for growing and harvesting bacteria (e.g., inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centrifuge with rotor and tubes or bottles for harvesting cells)

■ QIArack or equivalent holder (see “Setup of QIAGEN-tips”, page 13)

■ Ice

■ Isopropanol

■ 70% ethanol

■ Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)

For QIAGEN Plasmid Mini Kit protocol:

■ Microcentrifuge

■ 1.5 ml or 2 ml microcentrifuge tubes

For QIAGEN Plasmid Midi, Maxi, Mega, and Giga Kit protocols:

■ Centrifugation tubes or vessels with suitable capacity for the volumes specified in the appropriate protocol.

■ Refrigerated centrifuge capable of ≥20,000 x g with rotor for the appropriate centrifuge tubes or bottles

Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit

Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits

Retrieved from "http://2007.igem.org/wiki/index.php/Mini_and_Midi_prep"