EndoFree Plasmid Kits

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.

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.

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.

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

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

* 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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

LyseBlue reagent is provided in the following QIAGEN plasmid kits:

• QIAGEN Plasmid Kits (Mini-, Midi-, Maxi-, Mega and Giga)
• QIAfilter Plasmid Kits (Midi-, Maxi-, Mega and Giga)
• HiSpeed Plasmid Kits (Midi- and Maxi)
• EndoFree Plasmid Kit (Maxi-, Mega and Giga)
• QIAprep Spin Miniprep Kit
• QIAGEN Plasmid Plus Kits  (Midi, Maxi, Mega and Giga)