EndoFree Plasmid Kits

For purification of up to 10 mg endotoxin-free advanced transfection-grade plasmid or cosmid DNA

Features

  • Less than 0.1 EU/µg DNA achieved
  • Fast procedure, with integrated endotoxin removal
  • High yields of high-copy plasmid DNA
  • LyseBlue for optimum lysis and maximum DNA yield
EndoFree Plasmid Giga Kit (5)

Cat. No. / ID: 12391

5 QIAGEN-tip 10000, Reagents, 5 QIAfilter Mega-Giga Cartridges, Endotoxin-free Buffers
KitBuffer
EndoFree Plasmid Kit
EndoFree Plasmid Buffer Set
Cartridge type
Giga
Mega
Maxi
 
EndoFree Plasmid Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

EndoFree Plasmid Kits provide rapid, anion-exchange-based endotoxin-free plasmid DNA purification. QIAfilter Cartridges enable fast lysate clearing by filtration. The purified DNA exceeds the purity obtained by 2 x CsCl gradient centrifugation and is suitable for advanced transfection-grade applications. The EndoFree Plasmid Buffer Set can be used for preparations of 10 mega or 5 giga transfection-grade plasmid or cosmid DNA preparations.

Performance

The EndoFree Plasmid Kits integrate an efficient endotoxin removal step into the plasmid purification procedure — no additional extractions or affinity columns to remove lipopolysaccharides are required. Bacterial lysates are cleared by filtration with a QIAfilter Mega-Giga or Maxi Cartridge, and plasmid DNA is purified on gravity-flow QIAGEN-tips containing anion-exchange resin. Yields of up to 10 mg (giga), 2.5 mg (mega), and 500 μg (maxi) purified DNA are achieved from culture (culture volumes depend on plasmid copy number, size of insert, host strain, and culture medium). Purified DNA is endotoxin free (<0.1 EU/µg DNA).

For purification of low-copy plasmids and cosmids, the EndoFree Plasmid Mega Kit is a better choice than the EndoFree Giga Plasmid Kit, due to the large culture volumes required and the limited capacity of the QIAfilter Mega-Giga Cartridge.

EndoFree Plasmid Kits remove bacterial endotoxins which are released during the lysis step and influence transfection of DNA into primary cells and sensitive cultured cells. The endotoxin-free DNA obtained from the EndoFree Plasmid Kits is highly suited for reproducible and reliable results in transfection (see figures Plasmid purification method versus transfection efficiency and Plasmid purity versus transfection efficiency and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). QIAGEN ultrapure endotoxin-free DNA is also suitable for gene therapy research and other sensitive applications.

 
Endotoxin levels in plasmid preparations*
Plasmid preparation method Endotoxin
(EU/µg DNA)
Average transfection
efficiency
EndoFree Plasmid Kit 0.1 154%
QIAGEN Plasmid Kit 9.3 100%
2x CsCl 2.6 99%
Silica-gel slurry 1230.0 24%
* Host strain: DH5α plasmid: pRSVcat.
1 ng LPS = 1.8 EU. 
Calculated from data in Figure "Plasmid purification method versus transfection efficiency".
EndoFree DNA yields high transfection efficiencies with primary cells*
DNA purification method Percentage of transfected cells
EndoFree Plasmid Kit 21.0% ± 0.93
QIAGEN Plasmid Kit 8.1% ± 0.57
Silica-gel slurry 5.2% ± 0.74
* Primary rabbit gastric parietal cells were transfected with pEGFP-N2 (CLONTECH) prepared by the methods indicated. Transfections were performed using Effectene Transfection Reagent. The data represent the percentage of cells expressing GFP as determined by scoring the number of green fluorescent cells 48 hours post transfection. The transfection efficiencies represent the average from 6 to 9 replicate dishes from more than 2 different DNA preps for each purification method. (Data kindly provided by C. Chew and J. Parente, Department of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.)

Principle

The level of endotoxin contamination in purified plasmid DNA depends on the purification method used (see table "Endotoxin levels in plasmid preparations"). Silica-slurry–purified DNA exhibits extremely high endotoxin levels. QIAGEN, QIAfilter, and HiSpeed Plasmid Kits and 2x CsCl ultracentrifugation yield very pure DNA with relatively low levels of endotoxin. EndoFree Plasmid Kits include an integrated endotoxin-removal step to yield plasmid DNA containing <0.1 EU/µg plasmid DNA.

QIAfilter Cartridges, provided in QIAfilter, HiSpeed, and EndoFree Plasmid Kits, are special filter units designed to replace centrifugation following alkaline lysis of bacterial cells. QIAfilter Cartridges completely remove SDS precipitates and clear bacterial lysates in a fraction of the time needed for centrifugation, reducing plasmid-purification time by up to 1 hour. QIAfilter Mega-Giga Cartridges operate with house vacuum to efficiently clear even large volumes of bacterial lysate with minimal effort (please note that the bottle is not included in the kits). QIAfilter Maxi Cartridges have a syringe-format and lysates are cleared in a matter of seconds by pushing the liquid through the filter.

The unique anion-exchange resin in QIAGEN-tips is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. Prepacked QIAGEN-tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation. The entire QIAGEN plasmid purification system avoids the use of toxic substances such as phenol, chloroform, ethidium bromide, and CsCl, minimizing hazard both to the user and the environment.

Endotoxins, also known as lipopolysaccharides or LPS, are cell-membrane components of Gram-negative bacteria such as E. coli (see figure "Bacterial cell wall"). Endotoxins are released during the lysis step of plasmid purification and significantly reduce transfection efficiencies in endotoxin sensitive cell lines (see figures "Plasmid purification method versus transfection efficiency" and "Plasmid purity versus transfection efficiency" and tables "Endotoxin levels in plasmid preparations" and "EndoFree DNA yields high transfection efficiencies with primary cells"). Furthermore, endotoxins can influence the uptake of plasmid DNA in transfection experiments by competing with DNA for “free” transfection reagent. Endotoxins also induce nonspecific activation of immune responses in immune cells such as macrophages and B cells, which can lead to misinterpretation of transfection results. These responses include induced synthesis of proteins and lipids such as IL-1 and prostaglandin. Overall, endotoxins represent a noncontrollable variable in transfection experiment setup, influencing the outcome and reproducibility of results and making them difficult to compare and interpret. In gene therapy research, endotoxins can interfere by causing endotoxic-shock syndrome and activation of the complement cascade.

Specifications

Features
EndoFree Plasmid
Giga Kit 
EndoFree Plasmid
Mega Kit
EndoFree Plasmid
Maxi Kit
Applications Research on gene therapy, transfection of sensitive cells Research on gene therapy, transfection of sensitive cells Research on gene therapy, transfection of sensitive cells
Culture volume/starting material 2.5 liters culture volume 500 ml – 2.5 liters culture volume 100–250 ml culture volume
Plasmid type High-copy, low-copy, cosmid DNA High-copy, low-copy, cosmid DNA High-copy, low-copy, cosmid DNA
Processing Manual (gravity flow) Manual (gravity flow) Manual (gravity flow)
Sample per run 1 sample per run 1 sample per run  1 sample per run 
Time per run 310 min 220 min  150 min
Yield <10 mg <2.5 mg <500 µg
 

 

Procedure

Bacterial cells are lysed under alkaline conditions and the crude lysates are cleared using the QIAfilter Cartridge. At this stage, the Endotoxin Removal Buffer is added to the filtered lysate, which is incubated on ice. The cleared lysate is then loaded onto the anion-exchange tip where plasmid DNA selectively binds under appropriate low-salt and pH conditions. RNA, proteins, metabolites, and other low-molecular-weight impurities are removed by a medium-salt wash, and ultrapure plasmid DNA is eluted in high-salt buffer (see flowchart "QIAGEN Plasmid Kit procedures"). The DNA is concentrated and desalted by isopropanol precipitation and collected by centrifugation.

Applications

DNA purified with EndoFree Plasmid Kits is suitable for any sensitive application, including:

  • Transfection, including primary, sensitive, and suspension cells 
  • Gene therapy research
  • Gene silencing
  • Microinjection

Supporting data and figures

Resources

Supplementary Protocols (2)
Endotoxin-free DNA is essential for gene therapy research and will improve transfection into sensitive eukaryotic cells.
This protocol is for purification of up to 100 µg endotoxin-free plasmid DNA using QIAGEN-tip 100.

Publications

Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA.
Bates MK; Zhang G; Sebestyén MG; Neal ZC; Wolff JA; Herweijer H;
Biotechniques; 2006; 40 (2):199-208 2006 Feb PMID:16526410
Drebrin E2 is differentially expressed and phosphorylated in parietal cells in the gastric mucosa.
Chew CS; Okamoto CT; Chen X; Thomas R;
Am J Physiol Gastrointest Liver Physiol; 2005; 289 (2):G320-31 2005 Mar 24 PMID:15790763
EBV-induced gene 3 transcription is induced by TLR signaling in primary dendritic cells via NF-kappa B activation.
Wirtz S; Becker C; Fantini MC; Nieuwenhuis EE; Tubbe I; Galle PR; Schild HJ; Birkenbach M; Blumberg RS; Neurath MF;
J Immunol; 2005; 174 (5):2814-24 2005 Mar 1 PMID:15728491
Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.
Aliprantis AO; Yang RB; Mark MR; Suggett S; Devaux B; Radolf JD; Klimpel GR; Godowski P; Zychlinsky A;
Science; 1999; 285 (5428):736-9 1999 Jul 30 PMID:10426996

FAQ

What is the composition of buffer STE?

The composition of Buffer STE is:

  • 100 mM NaCl
  • 10 mM Tris-Cl, pH 8.0
  • 1 mM EDTA

Buffer STE is a DNA resuspension and storage buffer used in QIAGEN Plasmid Kits for plasmid purification and in some plasmid supplementary protocols. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -415
What is the advantage of running an analytical gel with fractions of my plasmid preparation?

Running fractions saved from each step in the plasmid preparation procedure on an agarose gel enables monitoring the performance of each crucial step in the protocol. If the plasmid DNA is of low yield or quality, the samples can be analyzed to determine at what stage of the purification procedure the difficulty occurred.

Aliquots can be taken from the cleared lysate and the flow-throughs as indicated in the relevant protocols, precipitated with isopropanol and resuspended in a small volume of TE buffer.

Please see the Troubleshooting Section of the QIAprep Miniprep Handbook and Appendix A of the QIAGEN Plasmid Purification Handbook for instructions, and a picture and legend explaining the typical results you may see. You can also access this information on our Plasmid Resource Pages.

FAQ ID -769
Which QIAGEN plasmid preparation kits will contain LyseBlue Reagent?

LyseBlue reagent is provided in the following QIAGEN plasmid kits:

FAQ ID -865
How do I know if my plasmid is a high- or low copy number type?

Find out which origin of replication your plasmid contains, and look at the table below for classification into high-copy or low-copy types. This table can also be found online at the QIAGEN Plasmid Resource Center in the section 'Growth of bacterial cultures; Plasmid Copy Number' . A way to determine experimentally if the copy number of your plasmid is high or low is to perform a miniprep. A high-copy plasmid should yield between 3-5 ug DNA per 1 ml LB culture, while a low-copy plasmid will yield between 0.2-1 ug DNA per ml of LB culture.

 

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.

FAQ ID -350
Can I eliminate RNase A from buffer P1 for my plasmid preparation to obtain RNase-free DNA for in-vitro transcription?
No, RNase A should not be omitted from buffer P1. It is required to prevent RNA contamination of the purified plasmid DNA. RNase A will not interfere with downstream in-vitro transcription experiments, since it will be efficiently removed during the plasmid purification procedures using QIAGEN Plasmid Kits.
FAQ ID -366
I left Buffer P1 at room temperature after addition of RNase A, what shall I do?

Buffer P1 with RNase A used in QIAGEN Plasmid Purification Kits should be fine at room temperature for a few days. We would expect the enzyme to have some residual activity. However, optimal results cannot be guaranteed after storage at room temperature. If you notice that RNase A activity is substantially reduced, you can add fresh RNase A to your buffer.

We recommend that Buffer P1 with RNase A be stored in the refrigerator (2-8°C). RNase A will be stable for 6 months under this condition.

FAQ ID -859
What is the composition of buffer QN?

The composition of Buffer QN is:

  • 1.6 M NaCl
  • 50 mM MOPS, pH 7.0
  • 15 % isopropanol (v/v)

Buffer QN is the elution buffer used in EndoFree Plasmid Kits for preparation of endotoxin-free plasmid DNA. Details on buffer preparation and storage are presented in Appendix C of the EndoFree Plasmid Purification Handbook.

FAQ ID -414
3299-Can Buffer ER from the Endofree Plasmid kits be used instead of the Buffer ETR in QIAGEN Plasmid Plus kits?

No, the two buffers cannot be interchanged.  The QIAGEN Plasmid Plus chemistry is not compatible with Buffer ER from the Endofree Plasmid kits.

Which QIAGEN kit do you recommend for purifying plasmid DNA suitable for transfection of sensitive cells?
We recommend our EndoFree Plasmid Kits to isolate plasmid DNA suitable for transfecting sensitive cells and primary cells. The Endofree Plasmid kits are designed to remove endotoxins (i.e. lipopolysaccharides that are part of the bacterial cell wall) and are generally advantageous in providing higher transfection efficiencies.
FAQ ID -1092
With LyseBlue reagent for lysis control, can I now process more bacterial culture and overload the columns?

No. The maximum culture volumes recommended for QIAGEN's plasmid preparation kits still apply, and should be strictly followed. LyseBlue reagent now allows the user to monitor potential problems (insufficient bacterial cell resuspension and lysis as a consequence of overloading) early in the plasmid preparation process.

FAQ ID -864
What is the recipe for 2x YT?
2x YT medium, per liter 16 g tryptone 10 g yeast extract 5 g NaCl Media Preparation and Bacteriological Tools. Edited by: Fred M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith, Kevin Struhl Current Protocols in Molecular Biology (1994), Section 1.1.3.
FAQ ID -213
What are the additional plasmid bands I see on my gel?

Open circular plasmid, resulting from single strand nicks, usually migrates slower in agarose gels and forms (faint) bands above the supercoiled plasmid DNA band. Sometimes an additional band of denatured supercoiled DNA migrates just below the supercoiled form. This form may result from prolonged alkaline lysis with Buffer P2 and is resistant to restriction digestion.

For a detailed description on how to run and interpret an analytical gel, please see Appendix A in the QIAGEN Plasmid Purification Handbook: "Agarose Gel Analysis of the Purification Procedure", or visit this link.

FAQ ID -1059
What can I do when the DNA pellet prepared with QIAGEN Plasmid Kits has been overdried?
Redissolve the DNA by warming the solution slightly, e.g. incubate it at 37°C, and allow more time for redissolving.
FAQ ID -572
Why is my plasmid DNA yield low?

Low yields of plasmid DNA can be caused by a number of different factors. The most common causes for low yield are poor culturing conditions and plasmid propagation, excessive amounts of starting material resulting in insufficient bacterial cell lysis and column overloading. When working with the anion-exchange based QIAGEN Plasmid Purification Kits, extra care is required during the isopropanol precipitation step, as the glassy DNA pellet may be difficult to see, and tends to be only loosely attached to the side of the tube.

We strongly recommend to review the information provided on our Plasmid Resource Page in the section 'Optimal results with QIAGEN plasmid kits', as it provides useful background information on growing bacterial cultures and general considerations for optimal results. It is also necessary to follow the instructions in the relevant protocols precisely to ensure the best plasmid yield and quality.

To determine at what stage of the procedure any problem occurred, save fractions from different steps of the purification procedure, and analyze by agarose gel electrophoresis. For a detailed description on how to run and interpret an analytical gel, please see Appendix A in the QIAGEN Plasmid Purification Handbook: "Agarose Gel Analysis of the Purification Procedure", or visit the QIAGEN Plasmid Resource Center.

 

 

FAQ ID -768
Why do I get genomic DNA contamination in my plasmid prep?

Too vigorous mixing of the bacterial lysate causes genomic DNA to appear in the eluate. The lysate must be handled gently after addition of buffers P2 and P3 to prevent shearing of chromosomal DNA. The culture volume needs to be reduced if the lysate is too viscous for gentle mixing.

Use of LyseBlue Reagent enables visualization of efficient bacterial cell resuspension as a prerequisite for complete lysis, thereby helping to avoid overloading of the columns and additional difficulties related to highly viscous lysates.

Additional information for successful plasmid preparations using QIAGEN's broad selection of Plasmid Kits can be found at our Plasmid Resource Center.

FAQ ID -353
What is the composition of buffer TE?

The composition of Buffer TE is:

  • 10 mM Tris·Cl, pH 8.0
  • 1 mM EDTA

Buffer TE is a commonly used DNA resuspension and storage buffer. It is supplied in QIAGEN's Endofree Plasmid Kits, and used for plasmid DNA resuspension in combination with other QIAGEN Plasmid Kits. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -416
What is the composition of Buffer ER? Is it available separately?
The composition of buffer ER is confidential. It is included in the EndoFree Plasmid Kits, and it is not sold separately.
FAQ ID -571
Do you have a protocol for the removal of endotoxins from already purified plasmid DNA?

Yes. Endotoxins can be removed from purified plasmid preparations by following the Supplementary Protocol 'Removal of endotoxins from purified plasmid DNA using the EndoFree Plasmid Maxi Kit' (QP12).

This protocol requires the EndoFree Plasmid Buffer Set, which can be purchased separately. Alternatively, the EndoFree Plasmid Maxi Kit, containing all necessary components, can be used.

The plasmid DNA is first treated with endotoxin removal buffer ER, and then applied to QIAGEN's anion-exchange tip. After performing a wash step, the plasmid DNA is eluted from the tip, followed by isopropanol precipitation and redissolving the DNA in a suitable volume of endotoxin-free buffer TE.

FAQ ID -500
I am seeing a precipitate after adding LyseBlue reagent to Buffer P1. What should I do about that?

A precipitate forming upon adding LyseBlue reagent to Buffer P1 is a normal observation. This precipitate will completely dissolve after addition of Buffer P2. Please be sure to shake Buffer P1 vigorously before use to completely resuspend LyseBlue particles.

FAQ ID -1045
What is the RNase A concentration and composition of Buffer P1?

The composition of Buffer P1 is:

  • 50 mM Tris·Cl, pH 8.0
  • 10 mM EDTA
  • 100 µg/ml RNase A

After RNase A addition, the buffer should be stored at 2–8°C.

Buffer P1 is the resuspension buffer used in a variety of QIAGEN kits for plasmid DNA purification. Details on buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -198
Can Buffers N3 and P3 be used interchangeably?
No. Although both Buffers N3 of the QIAprep Spin Miniprep and P3 of the QIAGEN Plasmid Kits perform the neutralization step in an alkaline lysis procedure, they are completely different. Buffer N3 contains a proprietary formula that sets up binding conditions for the QIAprep Miniprep column's silica-gel-membrane. Buffer P3 sets up binding conditions for QIAGEN anion-exchange columns. Our website explains QIAGEN's nucleic acid purification technologies in more detail.
FAQ ID -310
What is the composition of buffer P3?

The composition of Buffer P3 is:

  • 3.0 M potassium acetate, pH 5.5

Buffer P3 is the neutralization buffer used in QIAGEN's anion-exchange based Kits for plasmid preparation. Details of buffer preparation and storage are presented in Appendix B of the QIAGEN Plasmid Purification Handbook.

FAQ ID -418
How can I keep my centrifuge tubes endotoxin-free?

In order to avoid recontamination of plasmid DNA after initial endotoxin removal, we recommend using only new plasticware which is certified to be pyrogen- or endotoxin-free. Endotoxin-free or pyrogen-free plasticware can be obtained from many different suppliers. Endotoxins adhere strongly to glassware and are difficult to remove completely during washing. Standard laboratory autoclaving procedures have little or no effect on endotoxin levels. Moreover, if the autoclave has previously been used for bacteria, the glassware will become extensively contaminated with endotoxin molecules. Heating glassware at 180°C overnight is recommended to destroy any attached endotoxin molecules. For further reading on endotoxin removal, please refer to the scientific literature (e.g. Weary M. and Pearson F., 1988. A manufacturer's guide to depyrogenation. Biopharm 1, 22-29).

General information on endotoxins, which can be efficiently eliminated with QIAGEN's EndoFree Plasmid Kits, is available online at our Plasmid Resource Center.

FAQ ID -301
Is plasmid DNA purified with QIAGEN Plasmid Purification Kits suitable for in vitro transcription?

Plasmid preparations are free of any detectable proteins or other contaminants when purified using QIAGEN's anion-exchange kits according to the recommended protocols. DNA purified using QIAGEN Plasmid Kits, QIAfilter Plasmid Kits, or EndoFree Plasmid Kits gives excellent results with in-vitro transcription experiments.

Although a high level of RNase A is employed at the beginning of the procedure, it is removed efficiently by potassium dodecyl sulfate precipitation and subsequent washing with Buffer QC. It is possible, although not necessary, to omit RNase A from the procedure when purifying DNA for in vitro transcription. In this case, increasing the volume of Wash Buffer QC is recommended (e.g., for a Midi preparation on a QIAGEN-tip 100, use at least 2x 30 ml of Buffer QC instead of 2x 10 ml).

FAQ ID -1
What is the composition of buffer QC?

Buffer QC is the wash buffer used in QIAGEN Plasmid Kits for plasmid purification and in QIAGEN Blood & Cell Culture kits.

The composition of Buffer QC is:

  • 1.0 M NaCl
  • 50 mM MOPS, pH 7.0
  • 15% isopropanol (v/v)

 

To make 1 liter of solution, dissolve 58.44 g NaCl, 10.46 g MOPS (free acid) in 800 ml distilled water. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with distilled water. Store at 15–25°C.

FAQ ID -412
How can I prevent clogging of QIAfilter cartridges?
It is important to completely mix and lyse bacterial cells with plasmid Buffers P1 and P2. Incomplete mixing results in sticky or slimy areas of lysate, which will clog the filter matrix. It is recommended to completely resuspend cells in Buffer P1 and vigorously mix after addition of Buffer P2. Also mixing after Buffer P3 addition needs to be complete to allow fluffy precipitation of cell debris, which will float up. If the white debris does not float, dislodge it from the QIAfilter barrel wall (e.g. using a sterile pipette tip). Otherwise it will collect on the filter matrix and can lead to clogging. Use of LyseBlue reagent will help to achieve proper mixing results.
FAQ ID -1060
What is the composition of buffer QBT?

Buffer QBT is the equilibration buffer used in QIAGEN Plasmid Kits for plasmid purification and in QIAGEN Blood & Cell culture kits.

The composition of Buffer QBT is:

  • 750mM NaCl • 50 mM MOPS, pH 7.0
  • 15% isopropanol (v/v)
  • 0.15 % Triton® X-100 (v/v)

To make 1 liter of solution, dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml distilled water. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with distilled water. Store at 15–25°C

FAQ ID -411
When is chloramphenicol amplification of plasmids performed?

When low-copy-number plasmids containing the pMB1 or ColE1 origin of replication are prepared using QIAGEN Plasmid Purification Kits, plasmid DNA yields can be improved by adding chloramphenicol to the culture medium (170 mg/liter) to amplify copy numbers. For details and precautions on the use of chloramphenicol when culturing plasmids, please refer to standard manuals for cloning procedures (e.g., Molecular cloning : A laboratory manual / T. Maniatis, E.F. Fritsch, J. Sambrook).

Cultures of bacteria containing low-copy-number plasmids amplified in the presence of chloramphenicol should be treated as if they contain high-copy-number plasmids when choosing the appropriate culture volumes for the QIAGEN-tip to be used.

Note that copy numbers of the current generation of plasmids are so high that selective amplification in the presence of chloramphenicol is not necessary to achieve high yields. Please see FAQ 350 for origins of replication and copy numbers of various plasmids.

FAQ ID -3
Why would clumps occur following the addition of Buffer P2 when using LyseBlue Reagent in a plasmid preparation?

Clumps that occur after addition of Buffer P2 in a bacterial lysate containing LyseBlue reagent indicate poor resuspension of the bacterial cell pellet in Buffer P1. This handling error leads to inefficient cell lysis, and incomplete precipitation of SDS, cell debris, and genomic DNA. When resuspending the cell pellet, vortexing longer or resuspending the pellet by pipetting up and down can help.

If cells have been resuspended properly in P1, “brownish areas” after P2 addition just indicate poor mixing of P1 and P2. To overcome this, continue mixing the solution by inverting it gently until a homogeneous blue suspension is achieved.

FAQ ID -862