From 2007.igem.org

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

This protocol is designed for preparation of up to 20 μg of high-copy plasmid or cosmid DNA using the QIAGEN Plasmid Mini Kit. For additional protocols, such as for cosmid, low-copy-number plasmid, BACs, PACs, P1s, and double-stranded M13 replicative form purification, see the recommendations at www.qiagen.com/goto/plasmidinfo .

Important notes before starting

■ New users are advised to familiarize themselves with the detailed protocol provided in this handbook. In addition, extensive background information is provided on our plasmid resource page www.qiagen.com/goto/plasmidinfo . ■ Optional: Remove samples at the steps indicated with the symbol ☞ in order to monitor the procedure on an analytical gel (see page 41) Things to do before starting ■ Add the provided RNase A solution to Buffer P1 before use. Use one vial of RNase A (centrifuge briefly before use) per bottle of Buffer P1, to give a final concentration of 100 μg/ml. ■ Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37°C. ■ Pre-chill Buffer P3 at 4°C. ■ Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use one vial LyseBlue (centrifuge briefly before use) per bottle of Buffer P1 to achieve a 1:1000 dilution. 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.

Procedure

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm). Use a tube or flask with a volume of at least 4 times the volume of the culture. 2. Dilute the starter culture 1/500 to 1/1000 into 3 ml selective LB medium. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm). Use a flask or vessel with a volume of at least 4 times the volume of the culture. The culture should reach a cell density of approximately 3–4 x 109 cells per milliliter, which typically corresponds to a pellet wet weight of approximately 3 g/liter medium. 3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. ƒ If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C. 4. Resuspend the bacterial pellet in 0.3 ml of Buffer P1. Ensure that RNase A has been added to Buffer P1. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle before use to ensure LyseBlue particles are completely resuspended. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. 5. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at room temperature (15–25°C) for 5 min. Do not vortex, as this will result in shearing of genomic DNA. The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After use, the bottle containing Buffer P2 should be closed immediately to avoid acidification from CO2 in the air. 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. 6. Add 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 5 min. Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After addition of Buffer P3, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris, and KDS. The lysate should be mixed thoroughly to ensure even potassium dodecyl sulphate precipitation. If the mixture still appears viscous, more mixing is required to completely neutralize the solution. 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 suspension indicates that the SDS has been effectively precipitated. 7. Centrifuge at maximum speed in a microcentrifuge for 10 min. Remove supernatant containing plasmid DNA promptly. Before loading the centrifuge, the sample should be mixed again. Centrifugation should be performed at maximum speed in 1.5 ml or 2 ml microcentrifuge tubes (e.g., 10,000–13,000 rpm in a microcentrifuge). Maximum speed corresponds to 14,000–18,000 x g for most microcentrifuges. After centrifugation, the supernatant should be clear. If the supernatant is not clear, a second, shorter centrifugation should be carried out to avoid applying any suspended or particulate material to the column. Suspended material (which causes the sample to appear turbid) will clog the column and reduce or eliminate flow. ☞ Remove a 50 μl sample from the cleared lysate and save it for an analytical gel (sample 1). 8. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow. Place QIAGEN-tips into a QIArack over the waste tray or use the tip holders provided with each kit (see “Setup of QIAGEN-tips” page 13). Flow of buffer will begin automatically by reduction in surface tension due to the presence of detergent in the equilibration buffer. Allow the QIAGEN-tip to drain completely. QIAGEN-tips can be left unattended, since the flow of buffer will stop when the meniscus reaches the upper frit in the column. 9. Apply the supernatant from step 7 to the QIAGEN-tip 20 and allow it to enter the resin by gravity flow. The supernatant should be loaded onto the QIAGEN-tip promptly. If it is left too long and becomes cloudy due to further precipitation of protein, it must be centrifuged again before loading to prevent clogging of the QIAGEN-tip. ☞ Remove a 50 μl sample of the flow-through and save for an analytical gel (sample 2). 10. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow. ☞ Remove a 220 μl sample of the combined wash fractions and save for an analytical gel (sample 3). 11. Elute DNA with 0.8 ml Buffer QF. Collect the eluate in a 1.5 ml or 2 ml microcentrifuge tubes (not supplied). Note: For constructs larger than 45–50 kb, prewarming the elution buffer to 65°C may help to increase yield. ☞ Remove a 45 μl sample of the eluate and save for an analytical gel (sample 4). 12. Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥10,000 rpm for 30 min in a microcentrifuge. Carefully decant the supernatant. All solutions should be at room temperature in order to minimize salt precipitation. Isopropanol pellets have a glassy appearance and may be more difficult to see than the fluffy, salt-containing pellets that result from ethanol precipitation. Marking the outside of the tube before centrifugation allows the pellet to be easily located. Isopropanol pellets are also more loosely attached to the side of the tube, and care should be taken when removing the supernatant. 13. Wash DNA pellet with 1 ml of 70% ethanol and centrifuge at 10,000 rpm for 10 min. Carefully decant the supernatant without disturbing the pellet. The 70% ethanol removes precipitated salt and replaces isopropanol with the more volatile ethanol, making the DNA easier to redissolve. 14. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris·Cl, pH 8.5) Redissolve the DNA pellet by rinsing the walls to recover all the DNA. Pipetting the DNA up and down to promote resuspension may cause shearing and should be avoided. Overdrying the pellet will make the DNA difficult to redissolve. DNA dissolves best under slightly alkaline conditions; it does not easily dissolve in acidic buffers. Determination of yield To determine the yield, DNA concentration should be determined by both UV spectrophotometry at 260 nm and quantitative analysis on an agarose gel. For reliable spectrophotometric DNA quantification, A260 readings should lie between 0.1 and 1.0. Agarose gel analysis We recommend removing and saving aliquots during the purification procedure (samples 1–4). If the plasmid DNA is of low yield or quality, the samples can be analyzed by agarose gel electrophoresis to determine at what stage of the purification procedure the problem occurred

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

Procedure

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