品牌： Axygen

规格：250Kit

With the exception of the RNase A (after addition to Buffer S1), all reagents are stable for a period of at least 12 months from the date of receipt when stored under ambient conditions. Avoid exposure to direct sunlight or extremes in temperature. Buffer S2 contains SDS which may precipitate if exposed to cold temperatures. If this occurs, simply warm with a 37°C source and gently agitate to resuspend.

To preserve RNase activity, the RNase A is suspended in a solution containing a high concentration of ammonium sulfate. On occasion, a precipitate may form. If this occurs, the precipitate is easily dissolved in Buffer S1 and the RNase activity is unaffected. RNase A: 50 mg/ml. Stable at room temperature for up to 6 months. Recommend -20°C for long-term storage.If a precipitate is present,use an aliquot of Buffer S1 to resuspend and transfer to the Buffer S1 bottle.

Buffer S1: Resuspension buffer. Store at 4°C after addition of RNase A.

Buffer S2: Lysis buffer. Store at room temperature.

Buffer S3: Neutralization buffer. Store at room temperature.

Buffer W1: Wash buffer. Store at room temperature.

Buffer W2 concentrate: Desalting buffer. Before using the kit, add ethanol according to

instructions on the bottle label. Either 100% or 95% denatured ethanol can be

used. Store at room temperature.

Eluent: 2.5 mM Tris-HCl, pH 8.5. Store at room temperature.

Introduction

The AxyPrep Plasmid Miniprep Kit is based upon a modified SDS-alkaline lysis of bacterial cells in combination with selective binding of the plasmid DNA to a special Miniprep column. Each column has a binding capacity of at least 20 µg. The protocol provides a simple and reliable method to achieve the rapid isolation of highly purified plasmid DNA. The protocol has been optimized for bacterial cultures grown in LB (Luria-Bertani) broth, but can also be used for cultures grown in rich broths, such as LBG (LB + 2% glycerol) and 2×YT. TB (Terrific Broth) is not recommended for plasmid purification. Each column can process up to 4 ml of bacterial culture grown in LB or up to 2ml of culture grown in rich broth. The entire procedure can be completed within 20 minutes. The highly purified plasmid DNA is eluted in a small volume of Tris buffer eluent or deionized water and can be used immediately for many routine applications, such as DNA sequencing, restriction digestion, in vitro transcription, library screening, ligation and transformation.

Caution

Buffer S2 contains NaOH which is a caustic reagent. Buffer S3 and Buffer W1 contain chemical irritants. When working with these buffers, always wear suitable protective clothing such as safety glasses, laboratory coat and gloves. Be careful and avoid contact with eyes and skin. In the case of such contact, wash immediately with water. If necessary, seek medical assistance.

Equipment and consumables required

• Benchtop microcentrifuge capable of 12,000×g

• AxyVac vacuum manifold (catalog #AP-VM) or comparable model with luer-type fittings

• Vacuum source capable of –25-30 inches Hg

• Vacuum regulator

• 100% or 95% (denatured) ethanol

Preparation before experiment

1) Before using the kit, add the RNase A to Buffer S1. Mix well and store at 4°C.

Note: If a precipitate is present, use a small volume of Buffer S1 to resuspend the RNase A and then transfer to the Buffer S1 bottle.

2) Add the volume of ethanol specified on the bottle label to the Buffer W2 concentrate and mix well.

Either 100% or 95% (denatured) ethanol can be used.

3) Check Buffer S2 for precipitation before each use. If precipitation occurs, incubate at 37°C to dissolve the precipitate and then equilibrate to room temperature. After use, the bottle should be closed immediately in order to avoid neutralization of NaOH by CO2 in the air.

4) Pre-warming Eluent to 65°C may improve elution efficiency.