Endo-free Plasmid DNA Extraction Mini Kit

 Composition

*5 Preps of RNase A from the sample have been added to Buffer I.

Storage

1.        The RNaseA and Buffer ETR can be transported at room temperature. Please store the RNase A and Buffer ETR at 2-8℃ after receiving the products.

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

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

Introduction

This kit developed by alkaline lysis extraction plasmids and column purification of nucleic acids., suitable for extracting up to 20 μg of endotoxin-free high-purity plasmid DNA from 1~5 ml bacterial culture (LB medium), especially suitable for eukaryotic cell transfection, in vitro transcription and translation and other molecular biology experiments.

Equipment and Reagents to Be Supplied by User

1.        Absolute ethanol

2.        Endotoxin-free, clean 1.5 ml microcentrifuge tube with lid, pipette, and tips

3.        Disposable gloves, paper towels and protective supplies

4.        Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes).

5.        Vortexer

Preparation before use

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

2.      Prepare an ice bath and a water bath for 42℃.

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

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

5.      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 3-5 ml of bacteria cultured overnight and discard the medium. Add 250 μl Buffer I (RNaseA added) to fully suspend the bacterial pellet.

* The bacterial pellet can be suspended by vortexer or pipette several times. Fully suspended bacteria without no visible small bacterial clumps, otherwise it will seriously affect the final plasmid yield.

2.     Add 250 μl Buffer II and mix thoroughly by inverting 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.

* This step CANNOT be mixed with a vortexer, otherwise genomic DNA will be mixed in the final prepared plasmid.

* When the bacteria have lysed sufficiently, the solution should be thick and translucent; If the above effect is not achieved, it may be too many bacteria, and can increase the invert number to achieve the effect of full bacterial lysing.

* This step should NOT take more than 5 min.

3.     Add 125 μl Buffer N3 and gently invert the tube to mix thoroughly until all remaining blue precipitates disappear and change to a pale-yellow precipitate.

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

5.     Transfer the supernatant from Step 4 to a clean 1.5 ml microcentrifuge tube (not provided), add 125 μl Buffer ETR, mix thoroughly, and incubate in an ice bath for 10 min to make the solution transparent.

* Buffer ETR is viscous, please add Buffer ETR by using 1000 μl pipette tip.

6.     Place the 1.5 ml tube to a water bath incubate at 42℃ for 5 min to give the solution a cloudy appearance.

7.     Centrifuge at 13000 rpm for 3 min and endotoxins will collect in the pale-yellow lower phase at the bottom of the 1.5 ml tube.

8.     Transfer the supernatant from Step 7 to a clean 1.5 ml microcentrifuge tube (not provided), add 600 μl Buffer HB, and mix well.

* It is better to transfer less supernatant than to bring pale-yellow in the lower phase with endotoxin at the bottom.

9.     Transfer 600 μl mixture in Step 8 to a Spin Column (the Spin Column is placed in a 2 ml Collection Tube), close the lid and centrifuge at 12000 rpm for 30 sec.

10.  Discard the filtrate, place the Spin Column back into the 2 ml Collection Tube. Transfer all the remaining mixture in Step 8 to the Spin Column, close the lid and centrifuge at 12000 rpm for 30 sec.

11.  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 seconds.

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

12.  Repeat Step 11 once.

13.  Discard the filtrate, place the Spin Column back into the 2 ml Collection Tube and centrifuge at 14000 rpm for 1 min.

* If the centrifuge does not reach 14000 rpm, centrifuge at the full speed for 2 min.

* This step is to remove the residual ethanol in the Spin column. Please do not omit it, otherwise, the subsequent experimental effect may be affected by the residual ethanol in the extracted plasmid.

14.  Discard the 2 ml Collection Tube, place the Spin Column in a clean 1.5 ml microcentrifuge tube (not provided), add 50~100 µl Buffer E or endotoxin-free ultra-pure water in the Spin Column membrane, close the lid, incubate at room temperature for 1 min, and centrifuge at 12000 rpm for 30 sec.

15.  Discard the Spin Column, the eluted plasmid DNA could be immediately used in various molecular biology experiments or stored at -20℃ for later use.