One-Step Plasmid DNA Extraction Mini Kit

Composition

Storage

1.      Lysozyme and RNase A can transport at room temperature. Please store at 2-8℃ after receiving the product. Other reagents and items, if stored at room temperature (0~30℃), without showing any reduction in performance within 2 years.

2.      After adding RNase A to Lysozyme, it should be stored at 2~8℃ regardless of whether it is added to Buffer FL. It can be stably stored for 4 months.

3.      After adding RNase A and Lysozyme to Buffer FL, it should be stored at 2-8℃, which will not affect the lysing effect within 4 months.

Introduction

This kit uses a novel one-step enzyme lysis technology to rapidly purify high-quality plasmid DNA from 1-5 ml overnight E. coli bacterial cultures in just 6 min. The unique buffer formula combines the three steps of suspend bacteria, lyse and neutralization in SDS alkaline lysis into one step and is combined with advanced silica membrane adsorption technology, which can efficiently and specifically bind plasmid DNA, and effectively remove impurities such as protein and genomic DNA. The eluted high-quality plasmid DNA can be directly used in molecular biology experiments such as enzyme digestion, PCR, sequencing, and transformation.

Equipment and Reagents to Be Supplied by User

1.      Isopropanol

2.      1.5 ml and 2 ml microcentrifuge tubes

3.      Pipettes and tips

4.      Protective equipment such as latex gloves, disposable masks, and paper towels

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

6.      Vortexer

7.      Water bath maybe needed

Preparation Before Use

1.      Before use, transfer the RNase A to the tube containing the Lysozyme, invert to dissolve the Lysozyme, then transfer all of the solution to Buffer FL, and tick the box on the label to mark "RNase A/Lysozyme added".

2.      After adding the Lysozyme and RNase A, Buffer FL should be stored at 2~8℃. If Buffer FL is stored at 2~8℃ for more than 4 months, the lysozyme and RNase A should be re-added, otherwise the lysis performance of Buffer FL maybe reduced.

8.      Add isopropanol to Buffer FW2 according to the instructions on the label of the reagent bottle and tick the box on the label to mark " Isopropanol added".

Notes

1.      This kit is suitable for end A^- host bacteria. The end A⁺ host bacteria contain endogenous nuclease; enzyme digestion will lead to the degradation of plasmids. It is recommended to use Plasmid DNA Extraction Mini Kit（Cat. No. 1001050）to extract plasmids.

2.      If the plasmids are low-copy plasmids or plasmids larger than 15 kb, BAC/PAC DNA Extraction Midi Kit（Cat. No. 1013020）is recommended for better extraction.

Protocol

1.     Before use, put Buffer FL (check whether it has been added with Lysozyme and RNase A) on ice, incubate to 0~4℃.

* Ensure that Buffer FL is used at a low temperature, otherwise it will affect the subsequent lysing, resulting in low yield and purity.

2.     Collect 1-5 ml of overnight cultured bacteria in a 2 ml microcentrifuge tube  (not provided) by centrifuging at 12000 rpm for 1 min，discard the medium (if there is a lot of bacterial cultures, the bacterial pellet can be collected into a 2 ml microcentrifuge tube through multiple centrifuges).

* Do NOT use 1.5 ml microcentrifuge tube to collect bacteria. Bacterial pellet at the bottom of the 1.5 ml tube will be difficult to suspend in step 3, resulting in many small bacterial clumps that are difficult to lyse.

3.     Add 600 μl Buffer FL, immediately vortex for 30 sec, completely resuspend the bacterial pellet, and incubate at room temperature (15-25℃) for 3 min. After using Buffer FL, please return it to 2-8℃ in time for storage.

* After adding Buffer FL, it must be vortex immediately to suspend the bacterial pellet until no bacterial clumps are visible, thereby to increase yield and avoid genomic DNA contamination. If the bacteria are not suspended immediately after adding Buffer FL (the effect is equivalent to adding uncooled Buffer FL), the surface of the bacterial pellet will be lysed first, which will affect the full suspension of the bacterial pellet, resulting in many small bacterial clumps that are difficult to disperse.

* If the ambient temperature is lower than 15℃，incubate the lysate at 37℃ for 3min, or it will cause RNA contamination in the purified plasmid DNA.

* If the lysate is slightly turbidity and non-viscous, it is normal and will not affect the subsequent experiment.

4.     Transfer all the lysate to a Spin Column (the Spin Column is placed in a 2 ml Collection Tube), centrifuge at 12000 rpm for 30 sec and discard the filtrate.

5.     Place the Spin Column back into the 2 ml Collection Tube, add 600 µl Buffer FW2, centrifuge at 12000 rpm for 30 sec, discard the filtrate.

* Ensure that isopropanol has been added to Buffer FW2.

6.     Place the Spin Column back into the 2 ml Collection Tube and centrifuged at full speed for 1 min.

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

7.     Discard the 2 ml Collection Tube, place the Spin Column into a clean 1.5 ml microcentrifuge tube (not provided), add 50~100 μl Buffer E to the center of the Spin Column, and centrifuged at 12000 rpm for 30 sec.

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