QIAGEN Plasmid Mini User manual

Sample to Insight__

February 2021

QIAGEN® Plasmid Purification Handbook

QIAGEN Plasmid Mini, Midi, Maxi, Mega, and Giga Kits

For purification of ultrapure, transfection-grade plasmid DNA

QIAGEN Plasmid Purification Handbook 02/2021

Contents

Kit Contents ............................................................................................................... 3

Shipping and Storage ................................................................................................. 4

Intended Use .............................................................................................................. 4

Safety Information ....................................................................................................... 5

Quality Control ........................................................................................................... 5

Introduction ................................................................................................................ 6

Principle and procedure .................................................................................... 6

Equipment and Reagents to Be Supplied by User ............................................................ 8

Important Notes .......................................................................................................... 9

Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit .............. 17

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

.............................................................................................................................. 23

Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mega and Giga Kits

.............................................................................................................................. 30

Protocol: Very Low-Copy Plasmid/Cosmid Purification Using QIAGEN-tip 100 or

QIAGEN-tip 500 ...................................................................................................... 39

Troubleshooting Guide .............................................................................................. 47

Appendix A: Agarose Gel Analysis of the Purification Procedure .................................... 52

Appendix B: Composition of Buffers ............................................................................ 56

Ordering Information ................................................................................................ 59

Document Revision History ......................................................................................... 64

QIAGEN Plasmid Purification Handbook 02/2021

Kit Contents

QIAGEN Plasmid Kit Catalog no.

QIAGEN-tip 20 25 100 – – – – –

QIAGEN-tip 100 – – 25 100 – – –

QIAGEN-tip 500 – – – – 10 25 4 x 25

Buffer P1 20 ml 2 x 20 ml 110 ml 500 ml 110 ml 2 x 150 ml 8 x 150 ml

Buffer P2 20 ml 2 x 20 ml 110 ml 500 ml 110 ml 2 x 150 ml 8 x 150 ml

Buffer P3 20 ml 2 x 20 ml 110 ml 500 ml 110 ml 2 x 150 ml 8 x 150 ml

Buffer QBT 60 ml 2 x 60 ml 2 x 60 ml 500 ml 2 x 60 ml 2 x 200 ml 8 x 200 ml

Buffer QC 2 x 60 ml 2 x 240 ml 3 x 205 ml 5 x 500 ml 3 x 240 ml 4 x 500 ml 16 x 500 ml

Buffer QF 30 ml 140 ml 200 ml 4 x 140 ml 200 ml 510 ml 4 x 510 ml

RNase A* 2 mg 2 x 2 mg 11 mg 50 mg 11 mg 2 x 15 mg 8 x 15 mg

LyseBlue® 20 µl 2 x 20 µl 110 µl 500 µl 110 µl 2 x 150 µl 8 x 150 µl

Quick-Start Protocol 1 1 1 1 1 1 1

* Provided in a 10 mg/ml or 100 mg/ml solution.

QIAGEN Plasmid Kit Catalog no.

QIAGEN-tip 2500 5 25 – –

QIAGEN-tip 10000 – – 5 –

Buffer P1 2 x 150 ml 3 x 500 ml 3 x 250 ml 110 ml

Buffer P2 2 x 150 ml 3 x 500 ml 3 x 250 ml 110 ml

Buffer P3 2 x 150 ml 3 x 500 ml 3 x 250 ml 110 ml

Buffer QBT 200 ml 2 x 500 ml 2 x 200 ml 2 x 60 ml

Buffer QC 5 x 240 ml 11 x 500 ml 7 x 500 ml 3 x 240 ml

Buffer QF 200 ml 2 x 510 ml 510 ml 2 x 85 ml

RNase A* 2 x 15 mg 3 x 50 mg 3 x 25 mg 11 mg

LyseBlue 2 x 150 µl 3 x 500 µl 3 x 250 µl 110 µl

Quick-Start Protocol 1 1 1 1

* Provided in a 10 mg/ml or 100 mg/ml solution.

Mini (25) 12123

Mega (5) 12181

Mini (100) 12125

Midi (25) 12143

Mega (25) 12183

Midi (100) 12145

Maxi (10) 12162

Giga (5) 12191

Maxi (25) 12163

Plasmid Buffer Set 19046

Maxi (100) 12165

QIAGEN Plasmid Purification Handbook 02/2021

Shipping and Storage

QIAGEN-tips should be stored dry and at room temperature (15–25°C). Under these conditions, the components are stable for 2 years without showing any reduction in performance and quality, unless otherwise indicated on the label.

The QIAGEN Plasmid Kits should be stored at room temperature. After adding RNase A, Buffer P1 should be stored at 2–8°C. Under these conditions, the components are stable for 6 months without showing any reduction in performance and quality.

Other buffers and RNase A stock solution can be stored for 2 years at room temperature.

Intended Use

The QIAGEN Plasmid Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines.

QIAGEN Plasmid Purification Handbook 02/2021

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety, where you can find, view, and print the SDS for each QIAGEN kit and kit component.

Quality Control

In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of the QIAGEN Plasmid Kit is tested against predetermined specifications to ensure consistent product quality.

QIAGEN Plasmid Purification Handbook 02/2021

Introduction

The QIAGEN Plasmid Purification Kits are based on the remarkable selectivity of patented QIAGEN resin, allowing purification of ultrapure supercoiled plasmid DNA with high yields.

Anion-exchange–based QIAGEN-tips yield transfection-grade DNA, which is highly suited for use in a broad variety of demanding applications such as transfection, in vitro transcription and translation, and enzymatic modifications. QIAGEN offers the most comprehensive portfolio of tailored plasmid purification kits for any scale, throughput, or downstream application. Select the optimum kit for your requirements by visiting our online selection guide at www.qiagen.com/plasmidselectionguide. For transfection, QIAGEN also offers the

advanced PolyFect combined with the high-quality plasmid DNA obtained from QIAGEN, QIAfilter, HiSpeed and EndoFree page 59).

Principle and procedure

, SuperFect®, and Effectene® transfection reagents. These reagents,

Plasmid Kits, provide optimal transfection results (see “Ordering Information”,

QIAGEN plasmid purification protocols are based on a modified alkaline lysis procedure, followed by binding of plasmid DNA to QIAGEN resin under appropriate low-salt and pH conditions. RNA, proteins, dyes, and low-molecular–weight impurities are removed by a medium-salt wash. Plasmid DNA is eluted in a high-salt buffer and then concentrated and desalted by isopropanol precipitation.

Each disposable QIAGEN-tip packed with QIAGEN resin is designed to operate by gravity flow, reducing the amount of hands-on time required for the purification procedure.

QIAGEN Plasmid Purification Handbook 02/2021

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 safety data sheets (SDSs), 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 (cat. no. 19015) or equivalent holder (see “Setup of QIAGEN-tips”, page 14)  Ice  Isopropanol  Ethanol, 70%  Plasmid resuspension buffer (e.g., TE buffer, pH 8.0, or Tris·Cl, pH 8.5)

For the 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

centrifuge tubes or bottles

with a rotor for the appropriate

QIAGEN Plasmid Purification Handbook 02/2021

Important Notes

Please take a few moments to read this handbook carefully before beginning the DNA preparation. If QIAGEN Plasmid Purification Kits are new to you, please visit our plasmid resource page at www.qiagen.com/goto/plasmidinfo. Also be sure to read and follow the appropriate detailed protocol.

Plasmid size

Plasmids up to approximately 150 kb can be purified using QIAGEN plasmid purification protocols. Constructs larger than 45–50 kb, however, may exhibit somewhat reduced elution efficiencies. Prewarming the elution buffer to 65°C may help to increase the yield of large plasmids. For the isolation of large cosmid and plasmid DNA constructs, the QIAGEN LargeConstruct Kit is available (see “Ordering Information”, page 59).

Plasmid/cosmid copy number

Plasmids and cosmids vary in copy number, depending on the origin of replication they contain, their size, and the size of insert. The protocols in this handbook are grouped according to the copy number of the plasmid or cosmid to be purified.

High- and low-copy plasmids and cosmids should be purified using one of these protocols:

 “Protocol: Plasmid or Cosmid DNA Purification

Using QIAGEN Plasmid Mini Kit”, page 17

 “Protocol: Plasmid or Cosmid DNA Purification

Using QIAGEN Plasmid Midi and Maxi Kits”, page 23

 “Protocol: Plasmid or Cosmid DNA Purification

Using QIAGEN Plasmid Mega and Giga Kits”, page 30

QIAGEN Plasmid Purification Handbook 02/2021

Very low-copy plasmids and very low-copy cosmids (<10 copies per cell) should be purified using “Protocol: Very Low-Copy Plasmid/Cosmid Purification Using QIAGEN-tip 100 or QIAGEN-tip 500”, page 39, which uses extremely large culture volumes to obtain good yields.

For more details, visit our plasmid resource page at www.qiagen.com/goto/plasmidinfo.

Host strains

The strain used to propagate a plasmid can have a substantial influence on quality of the

purified DNA. Host strains such as DH1, DH5 QIAGEN protocols. The slower-growing strain XL1-Blue also yields DNA of very high quality.

Strain HB101 and its derivatives, such as TG1 and the JM100 series, contain large amounts of carbohydrates that are released during lysis and can inhibit enzyme activities if not completely removed. In addition, some strains, such as JM101, JM110, and HB101, have high levels of endonuclease activity and yield DNA of lower quality. If the quality of purified DNA is not as expected, a change of host strain should be considered. If difficulty is encountered with strains such as TG1 and Top10F, we recommend either reducing the amount of culture volume or doubling the volumes of Buffers P1, P2, and P3 to improve the ratio of biomass to lysis buffers for optimized lysis conditions.

α, and C600 yield high-quality DNA with

QIAGEN Plasmid Purification Handbook 02/2021

Table 1. Origins of replication and copy numbers of various plasmids and cosmids

DNA construct Origin of replication Copy number Classification

Plasmids

pUC vectors pMB1* 500–700 High copy

pBluescript® vectors ColE1 300–500 High copy

pGEM® vectors pMB1* 300–400 High copy

pTZ vectors pMB1* >1000 High copy

pBR322 and derivatives pMB1* 15–20 Low copy

pACYC and derivatives p15A 10–12 Low copy

pSC101 and derivatives pSC101 ~5 Very low copy

Cosmids

SuperCos pMB1 10–20 Low copy

pWE15 ColE1 10–20 Low copy

* The pMB1 origin of replication is closely related to that of ColE1 and falls in the same incompatibility group. The

high-copy plasmids listed here contain mutated versions of this origin.

Culture media

QIAGEN plasmid purification protocols are optimized for use with cultures grown in standard

Luria Bertani (LB) medium to a cell density of approximately 3–4 x 10 corresponds to a pellet wet weight of approximately 3 g/liter medium. Please note that a number of slightly different LB culture broths, containing different concentrations of NaCl, are commonly used. We recommend growing cultures in LB medium containing 10 g NaCl per liter (Table 2) to obtain the highest plasmid yields.

cells/ml, which typically

Rich media are not recommended for plasmid preparation with QIAGEN-tips. If rich media must be used, growth time must be optimized and culture volumes reduced. For more details, visit our plasmid resource page at www.qiagen.com/goto/plasmidinfo.

QIAGEN Plasmid Purification Handbook 02/2021

Table 2. Composition of Luria Bertani (LB)* medium

Contents Per liter

Tryptone 10 g

Yeast extract 5 g

NaCl 10 g

* See Appendix B, page 56, for preparation of LB medium.

Culture volume

Do not exceed the maximum recommended culture volumes given at the beginning of each protocol (and on the card inside the back cover of this handbook). Using larger culture volumes will lead to an increase in biomass and can affect the efficiency of alkaline lysis, leading to reduced yield and purity of the preparation.

The protocol for the QIAGEN Plasmid Kits is optimized for use with cultures grown in standard

LB medium, grown to a cell density of approximately 3–4 x 10 harvesting cultures after approximately 12–16 hours of growth, which typically is the transition from logarithmic into stationary growth phase. It is best to assess the cell density of the culture and, if that is too high, to reduce the culture volumes accordingly or increase the volumes of Buffers P1, P2, and P3. A high ratio of biomass to lysis buffers will result in poor lysis conditions and subsequently low DNA yield and purity. For determination of cell density, calibration of each individual spectrophotometer is required to facilitate accurate conversion of OD measurements into the number of cells per milliliter. This can be achieved by plating serial dilutions of a culture onto LB-agar plates in the absence of antibiotics. The counted colonies are used to calculate the number of cells per milliliter, which is then set in relation to the measured OD

values.

600

cells per ml. We advise

600

QIAGEN Plasmid Purification Handbook 02/2021

Capacity of QIAGEN-tips

QIAGEN-tips are available in a variety of sizes for preparation of as little as 20 µg or as much as 10 mg plasmid DNA (Figure 1). The maximum plasmid-binding capacities of the QIAGEN-tips 20, 100, 500, 2500, and 10000 are at least 20 µg, 100 µg, 500 µg, 2.5 mg, and 10 mg, respectively. Actual yields will depend on culture volume, culture medium, plasmid copy number, size of insert, and host strain. For more details, visit our plasmid resource page at www.qiagen.com/goto/plasmidinfo.

Figure 1. QIAGEN-tip 20 to QIAGEN-tip 10000.

QIAGEN Plasmid Purification Handbook 02/2021

Setup of QIAGEN-tips

QIAGEN-tips may be held upright in a suitable collection vessel such as a tube or flask, using the tip holders provided with the kits (Figure 2A). Alternatively, the QIAGEN-tips 20, 100, 500, and 2500 may be placed in the QIArack (cat. no. 19015) (Figure 2B).

Figure 2. Setup of QIAGEN-tips (A) with tip holder or (B) with the QIArack.

Optional if using the QIAfilter Mega-Giga Cartridge for lysate clearing:

Figure 3. The vacuum-operated QIAfilter Mega-Giga Cartridge in use. Note that the bottle is not included in kits.

QIAGEN Plasmid Purification Handbook 02/2021

Analytical gel analysis

The success of the plasmid purification procedure can be monitored on an analytical gel

Figure 4, page 53). We recommend removing and saving aliquots where indicated during

( the purification procedure (samples 1–4). If the plasmid DNA is of low yield or low quality, the samples can be analyzed by agarose gel electrophoresis to determine the stage of the purification where the problem occurred (see page 53).

Convenient stopping points in protocols

For all protocols, the purification procedure can be stopped and continued later by freezing the cell pellets obtained by centrifugation. The frozen cell pellets can be stored at

−30 to −15°C for several weeks. In addition, the DNA eluted from the QIAGEN-tip can be stored overnight at 2–8°C,* after which the protocol can be continued.

Using LyseBlue reagent

LyseBlue is a color indicator that provides visual identification of optimum buffer mixing. This prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris. This makes LyseBlue ideal for use by researchers who have not had much experience with plasmid preparations, as well as experienced scientists who want to be assured of maximum product yield.

LyseBlue can be added to the resuspension buffer (Buffer P1) bottle before use. Alternatively, smaller amounts of LyseBlue can be added to aliquots of Buffer P1, enabling single plasmid preparations incorporating visual lysis control to be performed.

LyseBlue reagent should be added to Buffer P1 at a ratio of 1:1000 to achieve the required working concentration (e.g., 10 µl LyseBlue into 10 ml Buffer P1). Make sufficient LyseBlue/Buffer P1 working solution for the number of plasmid preps being performed.

* Longer storage is not recommended.

QIAGEN Plasmid Purification Handbook 02/2021

LyseBlue precipitates after addition into Buffer P1. This precipitate will completely dissolve after addition of Buffer P2. Shake Buffer P1 before use to resuspend LyseBlue particles.

The plasmid preparation procedure is performed as usual. After addition of Buffer P2 to Buffer P1, the color of the suspension changes to blue. Mixing should result in a homogeneously colored suspension. If the suspension contains localized regions of colorless solution or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.

Upon addition of neutralization buffer (Buffer P3 or Buffer N3), LyseBlue turns colorless. The presence of a homogeneous solution with no traces of blue indicates that SDS from the lysis buffer has been effectively precipitated.

QIAGEN Plasmid Purification Handbook 02/2021

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-copynumber plasmid, BACs, PACs, P1s, and double-stranded M13 replicative form purification, see the recommendations at www.qiagen.com/goto/plasmidinfo.

Important points 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 indicated steps to monitor the procedure on an analytical

gel (see Appendix A, “Agarose

Things to do before starting

gel analysis

”, page 53).

 Before use, centrifuge RNase A briefly, and then add into Buffer P1 to obtain 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.

 Prechill Buffer P3 at 4°C.  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. For more details, see “Using LyseBlue reagent” on page 15.

QIAGEN Plasmid Purification Handbook 02/2021

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.

If you wish to stop the protocol and continue later, freeze the cell pellets at −30 to −15°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 for 5 min.

Do not vortex because 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

in the air.

QIAGEN Plasmid Purification Handbook 02/2021

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 the 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 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.

Optional: Remove a 50 µl sample from the cleared lysate and save it for an analytical gel (sample 1).

QIAGEN Plasmid Purification Handbook 02/2021

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 14). 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 because 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.

Optional: 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.

Optional: 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 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.

Optional: Remove a 45 µl sample of the eluate and save for an analytical gel (sample 4).

+ 46 hidden pages

QIAGEN Plasmid Mini User manual

Specifications and Main Features

Frequently Asked Questions

User Manual