QIAprep 96 Turbo Kits

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.

The average eluate volume when using either spin or vacuum protocols with the QIAprep 96 Turbo Miniprep Kit is 60 µl. Since 100 µl Buffer EB (10 mM Tris-Cl, pH 8.5) or water are added to each well for elution, the dead volume per sample is approximately 40 µl for both vacuum and spin procedures.

All QIAprep Miniprep Kits can be used for preparation of low-copy number plasmids and cosmids up to 50 kb. Below are recommendations for processing low-copy constructs using QIAprep technology:

• Use up to 10 ml overnight E. coli cultures grown in LB medium
• Be sure to include the optional Buffer PB wash step for all bacterial strains
• When plasmids or cosmids are larger than 10 kb, pre-heat Buffer EB (or water) to 70°C prior to eluting DNA from the QIAprep membrane
• When using 10 ml culture volume, it is recommended to double the volumes of Buffers P1, P2, and N3

See also QIAGEN News 1998, Issue 5 for an article entitled 'Isolation of a low-copy plasmid from agrobacterium using QIAprep technology'. Alternatively, the R.E.A.L. Prep 96 Plasmid Kit can be used for high-throughput purification of larger plasmids (e.g., BACs, PACs, and P1s). See QIAGEN News 1999, Issue 2 for an article entitled 'High-throughput purification of BACs with the new R.E.A.L. Prep 96 protocol'.

For high-throughput BioRobot plasmid isolation systems such as R.E.A.L. Prep 96, QIAwell Ultra, or QIAprep 96 Turbo Miniprep, there is no need for visual lysis control with LyseBlue reagent, because

1. insufficient resuspension and lysis have not been observed with these systems
2. automation systems already handle the lysis steps well enough
3. optical reading capabilities are not implemented on our BioRobot platforms

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.

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.

Yes, please follow the Supplementary Protocol 'Isolation of plasmid DNA (2x96) using the Sigma Centrifuge 4-15' (PR02).

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 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.

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.