Spin column-based bacterial DNA extraction is a widely used technique in molecular biology due to its speed, efficiency, and ease of use. This method utilizes a silica-based membrane within a spin column to selectively bind DNA in the presence of high salt concentrations.
Bacterial cells are initially lysed to release their contents, and the resulting lysate is applied to the spin column.
Contaminants such as proteins and RNA are removed through wash steps, and the purified DNA is finally eluted in a low-salt buffer or water.
This extracted DNA can then be used in downstream applications such as PCR, sequencing, and restriction digestion.
Benefits of Using Spin Columns:
- Fast and efficient
- High DNA yield and purity
- Easy to use and automate
- Harvesting and Lysis: Bacterial cells are collected and then lysed using a lysis buffer containing enzymes and detergents to break down the cell wall and membranes, releasing the DNA.
- Binding: The lysate is then added to a spin column containing a silica membrane. Under specific buffer conditions, the DNA binds to the silica membrane, while other cellular contaminants are washed away.
- Washing: The bound DNA is washed with wash buffers to remove any remaining impurities.
- Elution: Finally, the purified DNA is eluted from the silica membrane using an elution buffer or water.
Key Points:
- Specific reagents and buffers used may vary depending on the kit or protocol.
- Centrifugation is used to move the lysate and buffers through the spin column.
- The quality and quantity of the extracted DNA are crucial for downstream applications.
TE Buffer Ingredients:
- 10 mM Tris-HCl
- 1 mM EDTA
Lysis Buffer :
- Tris-Hcl (pH)-8.0 : 1M
- SDS (10%): 0.5-1%
- NaCl : 5M
- SDS :20%
Binding Buffer :
A binding buffer is a reagent used to bind DNA to silica during DNA extraction. It helps to purify DNA from various samples, including blood, cells, and tissues. It contains Guanidine Hydrochloride ,Tris-HCl and Triton X-100.
Washing buffer :
DNA Wash Buffer (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)
Elution buffer :
(0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0 )
Protocol :
- Transfer upto 2 ml overnight grown bacterial culture into a microfuge tube (1.5 ml ). Centrifuge at full speed (13,000 rpm) for 2 min .
- Discard medium completely and re-suspend the pellet in 180 μl TE Buffer or Elution Buffer. Centrifuge at full speed (5000 rpm) for 5 min and discard the supernatant
- Add 200μl of Lysis buffer and Mix thoroughly by vortexing.
- Incubate at 65°C for 15-20 minutes either on thermo mixer or water bath or dry bath. Briefly invert the tube once during incubation.
- After incubation add 100μl Isopropanol to the lysate. Mixed by inverting a tube 15- 20 times.
- Transfer entire lysate to the DNA spin column, and centrifuge at 12,000 rpm for 2 mins. Discard the flow through liquid .
- Repeat above step, until the entire sample has been processed and retain column for further processing.
- Place the column into the new collection tube. Add 500μl of binding buffer . Centrifuge at 12,000 rpm for 2 min. discard the flow through.
- Place the column into new collection tube. Add 500μl of Wash buffer. Centrifuge at 12,000 rpm for 1 min. discard the flow through.
- Place empty DNA spin column, with the lid open into the new collection tube and centrifuge at 12,000 rpm for 2 min.
- Place the column into a new sterile 1.5 ml eppendorf tube, add 30 μl preheated Elution buffer. Incubate at room temperature for 5 min. (perform this step twice 30 μl + 30 μl = 60 μl) (In this tube DNA will be eluted in 1.5 ml eppendrof tube so don’t discard it ).
- Centrifuge 10,000 rpm for 1 min to elute pure gDNA. The first elution normally yields 60-70 % of DNA bound. A second elution with another 30 μl buffer will yield another 20 % of the DNA.
- Note: Elution volume may vary as per downstream process
- Discard the Column, and save elute. Do not reuse binding columns or collection tubes.
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