GEL Purification: E.Z.N.A Gel Extraction Kit
Introduction
Materials
- Omega E.Z.N.A. Gel Etraction Kit
- Purifications
- HiBand DNA Mini Columns
- 2mL Collection Tubes
- Binding Bufer (XP2)
- Elutioon Buffer
- SPW Wash Buffer
Procedure
Spin Protocol
- Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments.
Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.
- Carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel.
Minimize the size of the gel slice by removing extra agarose.
- Determine the appropriate volume of the gel slice by weighing it in a clean 1.5mL microcentrifuge tube.
Assuming a density of 1g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3g will have a volume of 0.3mL.
- Add 1 volume Binding Buffer (XP2). IMPORTANT:
- Incubate at 60°C for 7 minutes or until the gel has completely melted.
Vortex or shake the tube every 2-3 minutes.
- In the meantime dilute SPW Wash Buffer with 100% ethanol as follows and store at room temperature: 100mL 100%Ethanol per bottle (for kit nb. D2500-02, the one we will be using)
- Insert a HiBind® DNA Mini Column in a 2mL Collection Tube.
- Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind® DNA Mini Column.
- Centrifuge at 10,000 x g for 1 minute at room temperature.
- Discard the filtrate and reuse collection tube.
- Repeat Steps 7-9 until all of the sample has been transferred to the column.
- Add 300 μL Binding Buffer (XP2).
- Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
- Discard the filtrate and reuse collection tube.
- Add 700 μL SPW Wash Buffer.
SPW Wash Buffer must be diluted with 100% ethanol prior to use.
- Centrifuge at maximum speed for 1 minute at room temperature.
- Discard the filtrate and reuse collection tube.
- Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
It is important to dry the HiBind® DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.
- Transfer the HiBind® DNA Mini Column to a clean 1.5mL microcentrifuge tube.
- Add 30-50 μL Elution Buffer or deionized water directly to the center of the column membrane.
The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent on pH. If eluting DNA with deionized water, make sure that the pH is around 8.5.
- Let sit at room temperature for 2 minutes.
- Centrifuge at maximum speed for 1 minute.
This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
- Store DNA at -20°C.
In an effort to purify the a PCR product from a gel, the E.Z.N.A. Gel Etraction Kit will be used. Link to the user manual: https://www.omegabiotek.com/wp-content/uploads/2018/07/D2500.D2501-PROTOCOL-E.Z.N.A.-Gel-Extraction-Kit.pdf