High Pure Plasmid DNA Extraction Mini Kit

Composition

* 5 Preps of RNaseA has been added to Buffer Ⅰ.

Storage

1.        Alkaline Protease can be transported at room temperature. After receiving the product, please store at -20℃.

2.        RNase A can be transported at room temperature. After receiving the product, please store RNase A at 2-8℃.

3.        After adding RNase A, Buffer Ⅰ should be stored at 2-8℃. If Buffer Ⅰ is stored for more than 6 months, RNase A should be re-added to the final concentration is 100 μg/ml.

4.        Other reagents and items, if stored at room temperature (0~30℃) , without showing any reduction in performance and would be stable within two years. If the product is stored at 2~8℃, the validity period of the product can be extended to more than two years (the product stored at 2~8℃ should be restored to room temperature before use).

Introduction

This kit combines the principle of extracting plasmid by alkaline lysis and column purification of nucleic acid technology, which is suitable for extracting up to 50 μg of high purity plasmid DNA from 1~5 ml of various wild type strains. This product is equipped with a special alkaline protease, which can efficiently hydrolyze and remove nuclease, so that the obtained plasmid is more pure and more stable. It is suitable for molecular biology experiments such as sequencing, in vitro transcription and translation, restriction enzyme digestion, and bacterial transformation.

Equipment and Reagents to Be Supplied by User

1.        Absolute ethanol

2.        1.5 ml centrifuge tube, pipette, and tips

3.        Disposable gloves, paper towels and protective supplies

4.        Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml Collection Tubes)

5.        Vortexer

Preparation before use

1.      If the centrifuge has a refrigeration function, set the temperature to 25℃.

2.      Add 1 ml Buffer Ⅰ to the tube containing RNase A, mix well, then transfer the solution back into the bottle containing Buffer Ⅰ. Mark "RNase A added" on the box of the label and store at 2-8℃.

3.      Add absolute ethanol to Buffer W2 according to the instructions on the label of the reagent bottle and tick the box of the label to mark "Ethanol added".

4.      When the room temperature is lower than 15℃, check Buffer Ⅱ before use for salt precipitation. Redissolve any precipitate by warming to 37°C.

Protocol

1.     Centrifuge at 12000 rpm for 30 sec to collect 1-5 ml overnight cultures of Escherichia coli in LB medium. Add 250 μl of Buffer Ⅰ (RNaseA added) to fully suspend the bacterial pellet.

* The bacterial pellet can be suspended by vortex or pipette several times. Fully suspended with no visible small bacterial clumps left, otherwise it will seriously affect the final plasmid DNA ‘yield.

* To extract plasmids from Gram-positive bacteria, add 20 μl of lysozyme solution with a concentration of 100 mg/ml at the end of this step, vortex to mix, and incubate at 37℃ for 10-30 min to digest the bacterial cell wall.

2.     Add 250 μl Buffer Ⅱ and gently invert the tube 4-6 times.

* Before using Buffer Ⅱ, ensure that there is no salt precipitation in the solution; After using Buffer Ⅱ, the bottle should be closed tightly to avoid long-term contact with air.

* Do not mix this step by vortexing, otherwise genomic DNA will be mixed in the final prepared plasmid DNA.

* When the bacteria have lysed sufficiently, the lysate should be thick and translucent; If the lystae does not reach the translucent effect, it may be too many bacteria were used, and the number of inverts must be increased to achieve the effect of full bacterial lysing.

3.     Add 10 µl of Alkaline Protease, gently invert 4-6 times, and incubate at room temperature for 5 min.

4.     Add 350 μl Buffer Ⅲ and gently invert the tube until the upper layer of light blue solution disappears and a light-yellow precipitate is formed.

* This step should not be mixed by vortexing, otherwise, genomic DNA will be mixed in the final prepared plasmid.

* When the action of this step is sufficient, there should be a loose yellowish flocculent precipitate, If the sediment appears thick, it may be too many bacteria were used, and the number of inverts can be increased to make the precipitation loose.

5.     Centrifuge at 13,000 rpm for 10 min.

6.     Place a Spin Column in a 2 ml Collection Tube, decanting the supernatant from step 5 into the Spin Column, close the lid and centrifuge at 12000 rpm for 30 sec.

7.     Discard the filtrate, place the Spin Column back into the 2 ml Collection Tube, add 500 µl Buffer W1 to the Spin Column, close the lid, centrifuge at 12000 rpm for 30 sec.

* The filtrate does not need to be completely discarded. To avoid contamination of the centrifuge by the filtrate adhering to the nozzle of the centrifuge tube, the 2 ml Collection Tube can be inverted and slapped once on a paper towel.

8.     Discard the filtrate, place the Spin Column back into the 2 ml Collection Tube, add 700 µl Buffer W2 to the Spin Column, close the lid and centrifuge at 12000 rpm for 30 sec.

* Ensure that absolute ethanol has been added to Buffer W2.

9.     Discard the filtrate, place the Spin Column back into the 2 ml Collection Tube and centrifuge at 14,000 rpm for 1 min.

* If the centrifuge does not reach 14,000 rpm, centrifuge at full speed for 2 min.

* This step is to remove the remaining ethanol. Please do not omit it. Otherwise, the effect of the subsequent experiment may be affected due to the residual ethanol in the purified plasmid.

10.  Discard the 2 ml Collection Tube, place the Spin Column in a clean 1.5 ml centrifuge tube. Add 50-100 µl Buffer E to the Spin Column membrane, close the lid, incubate at room temperature for 1 min and centrifuged at 12000 rpm for 30 sec.

* If the centrifuge does not have a leak-proof cover, change the centrifuge condition to 8000 rpm for 1 min, to avoid damage to the centrifuge by the 1.5ml centrifuge tube cap falling off.

* DNA can also be eluted with deionized water but ensure that the pH of the deionized water used is 7.0-8.5, otherwise it will affect the efficiency of DNA elution.

11.  Discard Spin Column, elution plasmid can be immediately used in various molecular biology experiments or store at -20℃ for later use.