13.7: Lab Technique - Isolation of DNA from Bacteria (The "Mini-Prep")

Isolation of DNA from Bacteria

The isolation of plasmid DNA starts with a re-suspension of the bacteria in a re-suspension solution, followed by the lysis of the bacterial cells in a lysis solution and precipitation of proteins using a neutralization solution. A description of each solution is given below. Because of the lysis solution, this lab technique is often called an alkaline lysis isolation. Beyond the neutralization step, the DNA can then be purified further using a wide variety of commercial kits (e.g. Qiagen Plasmid kit, Promega Wizard kit) or by a phenol:chloroform extraction step, followed by precipitation with ethanol. Commercial kits bind the DNA to a column comprised of a charged membrane (usually silica), allowing any contaminants to be washed out of the column. Commercial columns will use either gravity-fed or centrifugation "spin-columns" to apply the sample to the silica membrane of the column and wash away contaminants (Figure \(\PageIndex{1}\)). The DNA can then be eluted from the membrane using a small volume of Tris-EDTA (TE) buffer or nuclease-free water for additional downstream applications.

Figure \(\PageIndex{1}\): Columns for DNA isolation (A) Gravity-fed columns use gravity to flow the DNA sample through the silica membrane. (B) Spin columns use centrifugation to flow the DNA sample through the silica membrane. (Columns for DNA Isolation by Patricia Zuk, CC BY 4.0)

DNA isolation Buffers

• Re-suspension Buffer: a solution containing Tris, EDTA and RNase A
  • provides a stable salt solution for re-suspension of the bacterial pellet
  • the RNAse enzymatically removes contaminating RNA
• Lysis Buffer: a mixture of SDS and NaOH
  • the SDS solubilizes the lipid and protein components of the cell membrane, lysing the bacterial cells and releasing the DNA into the solution
  • the NaOH increases the pH of the solution, denaturing proteins and chromosomal DNA
  • short incubation time prevents denaturation of plasmid DNA
  • avoid vigorous mixing to prevent mixing of plasmid and chromosomal DNA
• Neutralization Buffer: a solution of potassium acetate
  • acidity of solution is designed to neutralize the alkaline lysis solution
  • high salt concentration precipitates proteins, chromosomal DNA (bound to the cell wall), cellular debris, and SDS
  • precipitation forms a white, fluffy precipitate that is easily removed through centrifugation
  • smaller plasmid DNA is renatured and remains in solution

Mini-Prep Isolation Protocols

The protocols given below represent some typical alkaline lysis isolation protocols. The first protocol uses reagents found in the biotech lab. The next two protocols are commercial kits from Qiagen. The first Qiagen protocol given below is a "mini-prep" protocol using a gravity-fed column to bind DNA. This column-based kit is designed to isolate small amounts of plasmid DNA (up to 20 µg; the "mini-prep" kit). However, larger amounts of DNA can be isolated using other kits (e.g. up to 10 mg; the "giga-kit"). All of these column-based DNA isolation kits use the same protocol but use different volumes of reagents and larger columns for DNA binding. The second Qiagen protocol below summarizes the steps of the QIAPrep Spin Mini-prep kit that uses spin columns for the isolation of small amounts of plasmid DNA (up to 20 µg). For each of these Qiagen protocols, the composition of the buffers used can be found in the manufacturer's handbook. However, the re-suspension, lysis, and neutralization buffers are similar to the ones used in the alkaline lysis protocol.

Lab Protocol: Alkaline Lysis "Mini-Prep"

Prior to starting the protocol, add RNase A to an aliquot of Resuspension Buffer. Store this buffer at 4°C until needed.

1. Select individual bacterial colonies and use them to inoculate 2 mL aliquots of a liquid bacterial broth (e.g., LB broth, nutrient broth), containing the appropriate selection agent (e.g. antibiotic).
2. Incubate the bacteria samples for 16 to 18 hours at 37°C with shaking at 200 to 250 rpm. If desired, the bacterial cultures can be stored for several days at 4°C.
3. Transfer 1.5 mL of each bacterial to labeled microfuge tubes and centrifuge in a benchtop micro-centrifuge tube for 5 minutes at room temperature at 5,000 rpm.
4. Aspirate the supernatant.
5. Re-suspend each bacterial pellet in 200 µL of Resuspension Buffer and vortex to completely re-suspend.
6. Lyse the cells by adding 200 µL of Lysis Buffer and gently invert 4 to 5 times. Do not vortex the sample.
7. Neutralize each sample by adding 200 µL of chilled Neutralization Buffer and gently invert 9 to 10 times. Incubate on ice for 5 minutes. Do not vortex the sample.
8. Invert each sample 4 to 5 times and centrifuge for 10 minutes at 4°C (or room temperature) at 14,000 rpm.
9. Transfer each supernatant to a new 1.5 mL microfuge tube. If the supernatant is not clear, transfer the supernatant to a new microfuge tube, and repeat the centrifugation step for an additional 5 minutes. Transfer the cleared supernatant to a new tube. The supernatant volume will be approximately 500 µL
10. Add an equal volume of (i.e. 500 µL) of phenol:chloroform (i.e., 250 µL phenol and 250 µL chloroform). The phenol should be saturated to ensure neutral pH and prevent damage to the DNA sample.
11. Gently invert to mix.
12. Centrifuge for 5 minutes at 14,000 rpm at room temperature.
13. Remove the top aqueous layer and place into a new 1.5 mL microfuge tube. The top layer should be approximately 500 µL. Be careful not to remove any of the phenol:chloroform layer.
14. Add two (2) volumes of ice-cold 95% ethanol to the aqueous layer (i.e., 1000 uL).
15. Vortex and place on ice for 30 minutes to precipitate the DNA.
16. Centrifuge at 14,000 rpm for 30 minutes at 4°C (or room temperature).
17. Pour off (i.e., decant) the supernatant.
18. Wash the pellet with 125 µL of ice-cold 70% ethanol.
19. Centrifuge at 14,000 rpm for 5 minutes at 4°C (or room temperature) and decant the supernatant.
20. Air dry the pellet.
21. Re-suspend the pellet in 20 µL of TE buffer or nuclease-free water.
22. Confirm DNA and determine the DNA yield by UV spectroscopy.

Alkaline Lysis Mini-Prep Reagents

Resuspension Buffer

• dissolve the following in 800 mL distilled water:
  • 6.06 g Tris base
  • 3.72 g EDTA-2H₂0
• adjust the pH to 8.0 with HCl
• add distilled water to 1 L
• store at room temperature
• when needed, remove an aliquot and add RNAse A to a concentration of 100 µg/ml
• after RNase A addition, store buffer at 4°C
• final concentration:
  • 50 mM Tris-Cl, pH 8.0
  • 10 mM EDTA
  • 100 µg/ml RNase A

Lysis Buffer

• combine the following:
  • 80 µL of 10N NaOH
  • 400 µL of 10% SDS
  • 3520 µL distilled water
• store at room temperature
• final concentration:
  • 0.2N NaOH
  • 1% SDS (w/v)

Neutralization Buffer

• dissolve the following in 500 mL distilled water:
  • 294.5 g potassium acetate
• adjust the pH to 5.5 with glacial acetic acid
• add distilled water to 1 L
• store at room temperature
• final concentration:
  • 3 M potassium acetate, pH 5.5

TE Buffer (Tris-EDTA Buffer)

• 1 mL of 1 M Tris base (10 mM final)
• 200 µL of 0.5 M EDTA (1 mM final)
• pH to 8.0 with 10 N HCl and adjust volume to 100 mL with distilled water
• store at room temperature

Protocol: Qiagen Column "Mini-Prep"

Prior to starting the protocol, add RNase A to an aliquot of Resuspension Buffer. Store at 4°C until needed. A workflow for this protocol can be seen in Figure \(\PageIndex{2}\).

1. Select individual bacterial colonies and use them to inoculate 2 mL aliquots of a liquid bacterial broth (e.g., LB broth, nutrient broth), containing the appropriate selection agent (e.g. antibiotic).
2. Incubate the bacteria samples for 16 to 18 hours at 37°C with shaking at 200 to 250 rpm. If desired, the bacterial cultures can be stored for several days at 4°C.
3. Transfer 1.5 mL of each bacterial to labeled microfuge tubes and centrifuge for 5 minutes at room temperature at 5,000 rpm.
4. Aspirate the supernatant.
5. Re-suspend each bacterial pellet in 300 uL of Resuspension Buffer P1 and vortex to completely re-suspend.
6. Lyse the cells by adding 300 uL of Lysis Buffer P2 and gently invert 4 to 5 times. Do not vortex the sample. Incubate at room temperature for 5 minutes.
7. Neutralize each sample by adding 300 uL of chilled Neutralization Buffer P3 and gently invert 9 to 10 times. Incubate on ice for 5 minutes. Do not vortex the sample.
8. Invert each sample 4 to 5 times and centrifuge for 10 minutes at 4°C (or room temperature) at 14,000 rpm.
9. Transfer each supernatant to a new 1.5 mL microfuge tube. If the supernatant is not clear, transfer the supernatant to a new microfuge tube, and repeat the centrifugation step for an additional 5 minutes. Transfer the cleared supernatant.
10. For each DNA sample, prepare a Qiagen-Tip 20 column with 1.0 mL QBT Buffer and allow to drain through the column. Discard flow-through.
11. Add the supernatant from step 9 to the column and allow to flow through the column. Retain the flow-through in a new tube until DNA isolation is confirmed.
12. Wash the column with 2.0 mL QC Buffer (wash buffer containing ethanol) and allow to completely drain through the column. Discard flow-through.
13. Repeat the wash step.
14. Elute the DNA with 800 uL of QF Buffer and allow the flow through to collect into a new 1.5 mL microfuge tube.
15. Precipitate the DNA by added 0.7 volume (i.e., 560 uL for 800 uL supernatant) of room temperature isopropanol. Vortex or mix thoroughly.
16. Centrifuge at 14,000 rpm for 10 minutes at room temperature.
17. Pour off (i.e., decant) the supernatant carefully. Isopropanol-precipitated DNA pellets are more loosely attached to the tube than ethanol-precipitated pellets.
18. Wash the pellet with 1000 µL of 70% ethanol.
19. Centrifuge at 14,000 rpm for 5 minutes at or room temperature and carefully decant the supernatant.
20. Air dry the pellet.
21. Re-suspend the pellet in 20 µL of TE buffer or nuclease-free water.
22. Confirm DNA and determine the DNA yield by UV spectroscopy.

Note: The absence of DNA at the end of this protocol may be due to the failure of the DNA to bind the column prepared at step 11. The flow-through kept at step 12 will contain the DNA. This DNA can be reapplied to a new column prepared by QBT buffer. If DNA is present at the end of the protocol, the flow-through from step 12 can be safely discarded.

Figure \(\PageIndex{2}\): The Mini-Prep workflow for DNA isolation using a gravity-fed column. Plasmid DNA can be isolated from bacterial cells using a two-day column-based protocol. On day 1, bacterial colonies are selected and used to inoculate growth media. The cultures are grown "overnight" (i.e., 16 to 18 hours) at 37°C (steps 1 to 3). On day 2, the bacterial cells are pelleted through centrifugation and the supernatant decanted (steps 4 and 5). The pellet is re-suspended in a Resuspension Buffer (step 6). The cells are lyzed using an alkaline Lysis Buffer (step 7). The sample is neutralized and protein contaminants precipitated with a Neutralization buffer (step 8). The sample is centrifuged to pellet the contaminants (step 10) and the supernatant is applied to a prepared column that will bind the DNA (step 11). Contaminants are washed from the column using a Wash Buffer (step 12) and the bound DNA eluted from the column with an Elution Buffer (step 13). The eluted DNA is precipitated using either ice-cold 95% ethanol or room temperature isopropanol (step 14), followed by high-speed centrifugation (step 15). The supernatant is decanted and the precipitated DNA pellet washed with 70% ethanol. The sample is centrifuged again and the ethanol decanted. The resulting DNA pellet can be air dried. Spin-column protocols will use steps 1 through 10, followed by application to a spin column that uses centrifugation steps to bind, wash, and elute the DNA. Following elution, spin column protocols do not require ethanol precipitation steps (steps 14 and 15) and the DNA is ready to use for downstream applications. (The Mini-Prep Protocol by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2025))

Qiagen Mini-Prep Reagents

Resuspension Buffer P1

• dissolve the following in 800 mL distilled water:
  • 6.06g Tris base
  • 3.72g EDTA-2H₂0
• adjust the pH to 8.0 with HCl
• add distilled water to 1 L
• store at room temperature
• when needed, remove an aliquot and add RNAse A to a concentration of 100 µg/ml
• after RNase A addition, store buffer at 4°C
• final concentration:
  • 50 mM Tris-Cl, pH 8.0
  • 10 mM EDTA
  • 100 µg/ml RNase A

Lysis Buffer P2

• combine the following:
  • 80 µL of 10N NaOH
  • 400 µL of 10% SDS
  • 3520 µL distilled water
• store at room temperature
• final concentration:
  • 0.2N NaOH
  • 1% SDS (w/v)

Neutralization Buffer P3

• dissolve the following in 500 mL distilled water:
  • 294.5g potassium acetate
• adjust the pH to 5.5 with glacial acetic acid
• add distilled water to 1 L
• store at room temperature
• final concentration:
  • 3 M potassium acetate, pH 5.5

QBT Buffer

• dissolve the following in 800 mL distilled water:
  • 43.83 g NaCl
  • 10.46 g MOPS (free acid)
• adjust the pH to 7.0 with NaOH
• add 150 ml pure isopropanol
• add 15 ml 10% Triton X-100 solution
• add distilled water to 1 L with distilled water
• store at 15–25°C
• final concentration:
  • 750 mM NaCl
  • 50 mM MOPS, pH 7.0
  • 15% isopropanol
  • 0.15% Triton X-100

QC Buffer

• dissolve the following in 800 mL distilled water:
  • 58.44 g NaCl
  • 10.46 g MOPS (free acid)
• adjust the pH to 7.0 with NaOH
• add 150 ml pure isopropanol
• add distilled water to 1 L with distilled water
• store at 15–25°C
• final concentration:
  • 1.0 M NaCl
  • 50 mM MOPS, pH 7.0
  • 15% isopropanol

QF Buffer

• dissolve the following in 800 mL distilled water:
  • 73.05 g NaCl
  • 6.06 g Tris base in 800 ml distilled water
• adjust the pH to 8.5 with HCl.
• add 150 ml pure isopropanol
• add distilled water to 1 L with distilled water
• store at 15–25°C
• final concentration:
  • 1.25 M NaCl
  • 50 mM Tris-Cl, pH 8
  • 15% isopropanol

Protocol: Qiagen Spin-Column "Mini-Prep"

Prior to starting the protocol, add RNase A to an aliquot of Resuspension Buffer. Store at 4°C until needed. Add 95% ethanol to the PE wash buffer per the manufacturer's instructions. Store at room temperature until needed. The Neutralization Buffer N3 in this protocol cannot be used interchangeably with other Neutralization buffers such as P3.

1. Select individual bacterial colonies and use them to inoculate 2 mL aliquots of a liquid bacterial broth (e.g., LB broth, nutrient broth), containing the appropriate selection agent (e.g. antibiotic).
2. Incubate the bacteria samples for 16 to 18 hours at 37°C with shaking at 200 to 250 rpm. If desired, the bacterial cultures can be stored for several days at 4°C.
3. Transfer 1.5 mL of each bacterial to labeled microfuge tubes and centrifuge for 5 minutes at room temperature at 14,000 rpm.
4. Aspirate the supernatant.
5. Re-suspend each bacterial pellet in 250 µL of Re-suspension Buffer P1 (with RNase A) and vortex to completely re-suspend.
6. Lyse the cells by adding 250 µLof Lysis Buffer P2 and gently invert 4 to 5 times. Do not vortex the sample.
7. Neutralize each sample by adding 350 µL of Neutralization Buffer N3 and gently invert 4 to 5 times. Do not vortex the sample.
8. Centrifuge at room temperature for 10 minutes at 14,000 rpm.
9. If the supernatant is not clear, transfer the supernatant to a new microfuge tube, and repeat the centrifugation step for an additional 5 minutes.
10. For each DNA sample, place a QIAPrep spin column into a 2 mL collection tube.
11. Add the supernatant from step 8 to the spin column and centrifuge at room temperature for 60 seconds at 14,000 rpm. Retain flow through until DNA isolation is confirmed.
12. Wash the column with 500 µL Wash Buffer PE (containing ethanol) and centrifuge at room temperature for 60 seconds at 14,000 rpm.
13. Wash the column with 750 µL Wash Buffer PE and centrifuge at room temperature for 60 seconds at 14,000 rpm.
14. Discard flow through and centrifuge at room temperature for an additional 60 seconds at 14,000 rpm to remove residual wash buffer. The flow through must be removed prior to this centrifugation step or residual wash buffer will not be removed during centrifugation.
15. Place the washed spin column into a clean 1.5 mL microfuge tube.
16. Elute the DNA with 50 µL of EB Buffer (10 mM Tris-Cl, pH 8.5) or 50 uL of nuclease-free water. Let column stand for 1 min. Centrifuge at room temperature for 60 seconds at 14,000 rpm. TE buffer may also be used for elution.
17. Confirm DNA within eluate and determine the DNA yield by UV spectroscopy.

Qiagen Spin-Column Mini-Prep Reagents

Resuspension Buffer P1

• dissolve the following in 800 mL distilled water:
  • 6.06g Tris base
  • 3.72g EDTA-2H₂0
• adjust the pH to 8.0 with HCl
• add distilled water to 1 L
• store at room temperature
• when needed, remove an aliquot and add RNAse A to a concentration of 100 µg/ml
• after RNase A addition, store buffer at 4°C
• final concentration:
  • 50 mM Tris-Cl, pH 8.0
  • 10 mM EDTA
  • 100 µg/ml RNase A

Lysis Buffer P2

• combine the following:
  • 80 µL of 10N NaOH
  • 400 µL of 10% SDS
  • 3520 µL distilled water
• store at room temperature
• final concentration:
  • 0.2N NaOH
  • 1% SDS (w/v)

Neutralization Buffer N3

• cannot be used interchangeably with Neutralization Buffer P3
• must be purchased from Qiagen

Wash Buffer PE

• must be purchased from Qiagen

Elution Buffer EB

• 10 mM Tris-Cl, pH 8.5