QIAprep Spin Miniprep Kit – 质粒纯化

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 EB is:

• 10 mM Tris-Cl, pH 8.5

Buffer EB is the elution buffer used in the QIAquick PCR, Gel Extraction, Nucleotide Removal Kits, and MinElute Kits for DNA cleanup, and the QIAprep Miniprep Kits for small-scale plasmid purification. The purified DNA can also be eluted in TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

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.

• Add 1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample
• Mix, and store at –20°C for at least 1 h to precipitate the DNA
• Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15–20 min
• Pour off the ethanol and wash the pellet twice with room-temperature 70% ethanol
• Allow the DNA pellet to air-dry
• resuspend the DNA in a suitable volume of sterile TE buffer or distilled water

QIAprep spin miniprep kit is based on silica extraction chemistry. Average endotoxin levels that we have observed for Silica gel slurry is around 1200 EU/µg DNA.

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.

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.

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.

Here is the protocol for preparing buffer MP:

1. Dissolve 3.3 g citric acid monohydrate in 3 ml high-purity water at room temperature (21°C) in a 10 ml tube.
2. Stir the solution at 200 rpm for 5 min.
3. Filter the solution through a 0.2 μm sterile filter using a syringe to give a final volume of 6 ml Buffer MP.

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.

Yes, it is possible to isolate plasmid DNA from mammalian cells using the QIAprep Spin Miniprep kit . The article in QIAGEN News 1995 No. 2, page 11, Isolation of plasmid DNA from mammalian cells using QIAprep kit, describes a procedure that requires only 30 minutes compared to the time-consuming and labor-intensive Hirt method. 

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.

Unfortunately, we do not have any compatibility data for using potassium phosphate-based buffers instead of TE or water for the elution of plasmid DNA from the spin columns of the QIAprep Spin Miniprep Kit.

However, below is a reference where cDNA was eluted from QIAquick PCR Purification Kit columns with potassium phosphate buffer (4 mM, pH 8.5), after replacing the wash buffer (PE) with 5 mM potassium phosphate (pH 8.5) containing 80% ethanol:

Wang HY, Malek RL, Kwitek AE, Greene AS, Luu TV, Behbahani B, Frank B, Quackenbush J, Lee NH. "Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays." Genome Biol. 2003, 4(1): R5. Epub 2003 Jan 6.

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.

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.

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)

Yes, please follow either of the User-Developed Protocols:

• 'Isolation of plasmid DNA from Bacillus subtilis using the QIAprep Spin Miniprep Kit' (PR01)
• 'Isolation of plasmid DNA from Bacillus subtilis using the QIAGEN Plasmid Midi Kit' (QP02)

Yes, please follow the User-Developed Protocol 'Isolation of plasmid DNA from Agrobacterium using the QIAprep Spin Miniprep Kit; spin procedure' (PR03s).