Endo-free Plasmid DNA Extraction Maxi Kit

Composition

Storage

1.        The RNase A 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 RNase A, 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℃), without showing significant change in performance 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 is developed by alkaline lysis extraction plasmid and nucleic acid column purification technology. It is suitable for extracting plasmid DNA from 120~250 ml bacterial culture (LB medium) up to 1.5 mg. It is suitable for molecular biology experiments such as sequencing, in vitro transcription and translation, digestion by restriction endonuclease, transformation, and eukaryotic transfection.

Equipment and Reagents to Be Supplied by User

1.      Absolute ethanol, isopropanol

2.      50 ml centrifuge tube, pipette, and tips

3.      Disposable gloves, paper towels and protective supplies

4.      Centrifuge(s) (with rotor for 50 ml centrifuge tubes)

5.      Vortexer, ice water bath, water bath, incubator

Preparation before use

1.      If the centrifuge has a refrigeration function, set the temperature to 30℃.

2.      Set the water bath to 42℃ and the incubator to 37℃.

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 of 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.        Collect 150~250 ml of overnight bacterial culture with a 50 ml centrifuge tube (if rich medium is used, the bacterial culture volume should be halved or less), discard the supernatant, place the centrifuge tube upside down on a paper towel for 1 min.

2.        Suspend the bacterial pellet in 10 ml of Buffer Ⅰ with RNaseA was added.

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

3.        Add 10 ml Buffer Ⅱ, gently invert 6-8 times to mix evenly and lyse the bacteria.

* Ensure that no salt precipitation in the solution before using Buffer Ⅱ; Cap the bottle tightly after using Buffer Ⅱ 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 due to the excessive number of bacteria and can increase the invert number to lyse sufficiently. Note that this step should not take more than 5 min.

4.        Add 5 ml Buffer N3 and gently invert the tube until all remaining blue precipitates in the solution have changed to pale-yellow precipitates. Centrifuge ≥12,000×g for 5 min.

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

5.        Place the Filter into a clean 50 ml centrifuge tube (not provided), draw out the Plunger, pour all the supernatant from step 4 into the filter, insert and gently push the Plunger so that all the filtrate drops into the 50 ml centrifuge tube.

* The supernatant may drip directly after being added to the filter, be careful to always keep the filter in the 50 ml centrifuge tube.

* The supernatant poured into the filter mixed with some yellow precipitate will not affect the filtration effect.

6.        Add 5 ml Buffer ETR to the filtrate, cover tightly, invert to mix, incubate at ice bath for 10-15 min. Invert mixing several times during the ice bath to make the mixture transparent.

7.        Incubate at 42℃ for 5 min, so that the mixture becomes turbid, ≥12,000×g centrifuge for 10 min.

* The centrifuge temperature must be set at 30℃, and if it is not found that the phase separation, it can be centrifuged again for 15 min.

* If a small yellow liquid drop is found suspended in the supernatant when the centrifuge is removed at the end of centrifuge, it may be brought up by the fluctuation when the centrifuge stops rotating, and it can be left for about 1 min.

8.        Transfer the supernatant to a clean 50 ml centrifuge tube without the yellow deposits containing endotoxins at the bottom.

9.        Add 14 ml isopropanol to the supernatant, close the lid tightly, and gently invert 10 times to mix well. Transfer 20 ml of the mixture into a Spin Column, cover the lid, and centrifuge ≥12,000×g for 1 min.

10.     Discard the filtrate, place the Spin Column back into the 50 ml centrifuge tube, transfer all the remaining mixture to the Spin Column, close the lid, centrifuge ≥12,000×g for 1 min.

11.     Discard the filtrate, place the Spin Column back into the 50 ml centrifuge tube. Add 10 ml Buffer W2 to the Spin Column, close the lid and centrifuge ≥12,000×g for 1 min.

* 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 50 ml centrifuge tube, and centrifuge ≥12,000×g for 5 min.

14.     Discard the 50 ml centrifuge tube and place the Spin Column in a clean 50 ml centrifuge tube (not provided). Open the lid and incubate at 37℃ for 10-15 min.

* This step is to remove the residual ethanol on the Spin Column. If the taste of ethanol can still be smelled in the Spin Column after this step is finished, the incubate time should be extended appropriately.

15.     Add 2 ml Buffer E into the Spin Column membrane, open the lid, stand for 5 min at room temperature, close the lid and centrifuge at full speed for 5 min.

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

*  If the concentration of the eluted DNA is more than 200 ng/μl, it is recommended to elute the Spin column once more to improve DNA recovery: transfer the DNA solution from step 15 to a container, place the Spin Column back into the 50 ml centrifuge tube, add 1 ml Buffer E to the Spin Column membrane. Open the lid, stand for 5 min. Close the lid and centrifuge at ≥12,000×g for 5 min.

16.     Discard the Spin Column, store the elute plasmid DNA at -20℃ for later use.