QIAfilter Plasmid Giga Kit

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.

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.

The composition of Buffer P2 is:

• 200 mM NaOH
• 1% SDS (w/v)

It should be stored at room temperature. Buffer P2 is the lysis 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.

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.

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.

If low-copy plasmids are used with QIAfilter Plasmid Kits, the cell lysates of two QIAfilter Cartridges can be filtered into one previously equilibrated QIAGEN-tip, resulting in doubled DNA yields.

Similarly, if low copy plasmids are used with HiSpeed Plasmid Kits, the lysates of two QIAfilter Cartridges can be filtered into one equilibrated HiSpeed Tip.

When using the silica-based QIAprep Spin Miniprep Kit, a protocol is contained in the QIAprep Miniprep Handbook, in Appendix C: Special Applications. The protocol is called: 'Purification of plasmid DNA prepared by other methods'.

For our anion-exchange based Plasmid Purification Kits, a protocol can be accessed online at our Plasmid Resource Center, and is called 'Re-Purification of Plasmid DNA Prepared by Methods other than QIAGEN Tips'.

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 FWB2 is:

• 1 M potassium acetate, pH 5.0

Buffer FWB2 is the QIAfilter wash buffer used in QIAfilter Plasmid Kits for plasmid purification. Details on buffer preparation and storage are presented in Appendix B of the QIAfilter Plasmid Purification Handbook.

If cell clumps are present after adding Buffer P2 to your samples when using a QIAGEN Plasmid Purification Kit in combination with LyseBlue Reagent, you have two options:

• pipet the cell clumps up and down for resuspension
• transfer any clumps to a separate tube, add Buffer P1 and mix vigorously for resuspension, add Buffer P2 for lysis, and subsequently transfer the lysed material back to combine it with the rest of the original solution

Note: Avoid incubation times longer than 5 minutes in Buffer P2 as this will irreversibly denature plasmid DNA. In the scenario above, Buffer P3 may need to be added to portions of the sample, which can be subsequently combined once resuspension, lysis and neutralization of all fractions is complete.

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.

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.

The components of the SOC medium are:

• 0.5% Yeast Extract
• 2% Tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 10 mM MgCl2
• 10 mM MgSO4
• 20 mM Glucose*

*Note: add Glucose after autoclaving the solution with the remaining ingredients, and letting it cool down. Sterilize the final solution by passing it through a 0.2 µm filter.

SOC medium can be stored at room temperature and is stable for several years.

White insoluble material in the resuspended plasmid DNA pellet indicates carry-over of salts and/or carbohydrates. Ensure that isopropanol is used at room temperature for precipitation. Some bacterial strains, such as TG1 and JM100, naturally produce a high level of carbohydrates. However, carbohydrate contamination may also be observed when using other strains. The most common cause of this problem is over-growth of bacterial cultures. To avoid this, closely follow the guidelines for Plasmid DNA Preparation in the Handbook that was provided with the respective QIAGEN Plasmid Kit.

Useful hints and information on optimizing plasmid preparations can be found at the QIAGEN Plasmid Resource Center.

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).

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.

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

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.

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.