TITLE: Quick Protocol for Extraction and Purification of Genomic DNA
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Genomic DNA Purification Consists of Two Stages:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Part 1: Sample Lysis</li><li style = "counter-reset:ol0;list-style-type:disc;">Part 2: Genomic DNA Binding and Elution</li></ul></div></div>

[STEPS]
?. [PART 1: SAMPLE LYSIS]
Please select the sample material that you want to use:Cultured CellsMammalian Whole Blood (non-nucleated)Nucleated Whole Blood (birds, reptiles)Animal Tissue
?. [PART 1: SAMPLE LYSIS]
Start with a cell pellet containing 1 x 104 – 5 x 106 cells (typical starting amount is 1 x 106 cells).Frozen cells: thaw cell pellet slowly on ice and loosen by flicking the tube several times. Resuspend in  cold PBS by pipetting up and down.Fresh cells: pellet by centrifugation at  for  and resuspend in  cold PBS by pipetting up and down.Ensure pellet is resuspended completely. If using lower cell inputs, the use of carrier RNA may be beneficial; see product manual for details.
100 µl
Centrifuge: 1000 34
100 µl
?. [PART 1: SAMPLE LYSIS]
Add  Proteinase K and  RNase A to the resuspended pellet and mix by vortexing briefly to ensure the enzymes are efficiently dispersed.
1 µl
3 µl
Do not add the enzymes and the Cell Lysis Buffer simultaneously, as the high viscosity of the lysate will prevent equal distribution of the enzymes. Addition of RNase A can be omitted if a low percentage of co-purified RNA will not affect downstream applications.
?. [PART 1: SAMPLE LYSIS]
Add  Cell Lysis Buffer and vortex immediately and thoroughly. The solution will rapidly become viscous.
100 µl
?. [PART 1: SAMPLE LYSIS]
Incubate for  at  in a thermal mixer with agitation at full speed (~ ).
56 °C
Centrifuge: 1400 33
Incubation for longer than 5 minutes is not necessary, but will not negatively affect the quality of the purified gDNA. If a thermal mixer is not available, use a heating block and vortex occasionally.
?. [PART 1: SAMPLE LYSIS]
Transfer  of whole blood to a 1.5 ml microfuge tube. If processing less than 100 μl of blood, add cold PBS to bring the total volume to 100 μl. For pre-aliquoted frozen samples, do not thaw; add Proteinase K, RNase A and Blood Lysis Buffer to the frozen sample in step 3.
100 µl
?. [PART 1: SAMPLE LYSIS]
Add  Proteinase K,  RNase A and  of Blood Lysis Buffer. Mix immediately by vortexing.
10 µl
3 µl
100 µl
For frozen samples, do not thaw; add reagents and lysis buffer to frozen sample and immediately proceed to step 4. When working with multiple samples, prepare a master mix of the three reagents to save pipetting steps. Addition of RNase A can be omitted if a low percentage of co-purified RNA will not affect downstream applications.
?. [PART 1: SAMPLE LYSIS]
Incubate for  at  in a thermal mixer with agitation at full speed (~ ).
56 °C
Centrifuge: 1400 33
Incubation for longer than 5 minutes is not necessary, but will not negatively affect the quality of the purified gDNA from most species. If a thermal mixer is not available, use a heating block and vortex occasionally.
?. [PART 1: SAMPLE LYSIS]
Transfer  whole blood to a 1.5 ml microfuge tube.
10 µl
?. [PART 1: SAMPLE LYSIS]
Add  cold PBS and mix by vortexing.
90 µl
?. [PART 1: SAMPLE LYSIS]
Add  Proteinase K and  RNase A, and mix again by vortexing.
10 µl
3 µl
Do not add the enzymes and the Blood Lysis Buffer simultaneously, as the high viscosity of the lysate will prevent equal distribution of the enzymes. Addition of RNase A can be omitted if a low percentage of co-purified RNA will not affect downstream applications.
?. [PART 1: SAMPLE LYSIS]
Add  Blood Lysis Buffer and vortex thoroughly. The solution will rapidly become viscous.
100 µl
?. [PART 1: SAMPLE LYSIS]
Incubate for  at  in a thermal mixer with agitation at full speed (~ ).
56 °C
Centrifuge: 1400 33
Incubation for longer than 5 minutes is not necessary, but will not negatively affect the quality of the purified gDNA.
?. [PART 1: SAMPLE LYSIS]
Cut tissue into small pieces to ensure rapid lysis and high yields.
?. [PART 1: SAMPLE LYSIS]
Add Proteinase K (according to the table below) and  of Tissue Lysis Buffer to each sample. Mix immediately by vortexing.   AB1TISSUE TYPEPROTEINASE K AMOUNT2Brain, Kidney, Skin, Ear Clips3 μl3All other tissues10 μl
200 µl
Ensure tissue particles are able to move freely in the lysis mix and do not remain stuck on the bottom of the tube. When working with multiple samples, prepare a master mix of Tissue Lysis Buffer and Proteinase K to save pipetting steps.
AB1TISSUE TYPEPROTEINASE K AMOUNT2Brain, Kidney, Skin, Ear Clips3 μl3All other tissues10 μl
?. [PART 1: SAMPLE LYSIS]
Incubate at  in a thermal mixer with agitation at full speed () until tissue pieces have completely dissolved (typically  – ).
56 °C
Centrifuge: 1400 33
If time is not limiting, additional incubation up to 3 hours can further improve yields and decrease residual RNA. If an incubator with agitation is not available, use a tube rotator placed within an incubator, shaking water bath or a heating block (vortex samples every 5-15 minutes to speed up lysis).
?. [PART 1: SAMPLE LYSIS]
Note: The following step can be omitted when working with fresh or frozen tissue amounts Centrifuge for maximum speed  to pellet debris. Transfer the supernatant to a fresh microfuge tube.
Centrifuge: 12.000 34, 3 min
This prevents residual debris from clogging the membrane binding sites and helps to reach maximal yield and purity. It is especially important to perform this step if sample appears turbid, contains residual particles, when working with stabilized tissue, or when working with brain or fibrous tissues.
?. [PART 1: SAMPLE LYSIS]
Add  of RNase A to the lysate, vortex thoroughly, and incubate for a minimum of  at  with agitation at full speed.
3 µl
56 °C
This step can be skipped if a low percentage of co-purified RNA will not affect downstream applications.
?. [PART 1: SAMPLE LYSIS]
Proceed to step 1 of Part 2: Genomic DNA Binding and Elution
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for   – .
400 µl
Thorough mixing is essential for optimal results.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer the lysate/binding buffer mix (~ ) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Proceed immediately to step 8.
600 µl
Avoid touching the upper column area with lysate/binding mix and avoid transferring foam that may have formed during lysis. Any material that touches the upper area of the column, including any foam, may lead to salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and centrifuge: first for  at  to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for  at maximum speed (> ) to clear the membrane. Discard the flow-through and the collection tube.
Centrifuge: 1000 34
Centrifuge: 12000 34
For optimal results, ensure that the spin column is placed in the centrifuge in the same orientation at each spin step (for example, always with the hinge pointing to the outside of the centrifuge); ensuring the liquid follows the same path through the membrane for binding and elution can slightly improve yield and consistency.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer column to a new collection tube and add  gDNA Wash Buffer.
500 µl
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Reinsert the column into the collection tube. Add  gDNA Wash Buffer and close the cap. Centrifuge immediately for  at maximum speed (> ), then discard the collection tube and flow through.
500 µl
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included).
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Centrifuge for  at maximum speed (> ) to elute the gDNA.
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and invert a few times, so that the wash buffer  reaches the cap. Centrifuge immediately for  at maximum speed (), and discard the flow through. The collection tube can be tapped on a paper towel to remove any residual buffer before reusing it in the next step.
Centrifuge: 12000 34
Inverting the spin column containing wash buffer prevents salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  –  preheated () gDNA Elution Buffer, close the cap and incubate at  for .
35 µl
100 µl
60 °C
60 Room temperature
Elution in 100 μl is recommended, but smaller volumes can be used and will result in more concentrated DNA but a reduced yield (20 – 25 % reduction when using 35 μl). Eluting with preheated elution buffer will increase yields by ~ 20 – 40 % and eliminates the need for a second elution. For applications in which a high DNA concentration is required, using a small elution volume and then eluting again with the eluate may increase yield (~ 10 %). The elution buffer (10 mM Tris-Cl, pH 9.0, 0.1 mM EDTA) offers strong protection against enzymatic degradation and is optimal for long term storage of DNA. However, other low-salt buffers or nuclease-free water can be used if preferred. For more details on optimizing elution, please refer to “Considerations for Elution & Storage” in the product manual.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for   – .
400 µl
Thorough mixing is essential for optimal results.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer the lysate/binding buffer mix (~ ) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Proceed immediately to step 8.
600 µl
Avoid touching the upper column area with lysate/binding mix and avoid transferring foam that may have formed during lysis. Any material that touches the upper area of the column, including any foam, may lead to salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and centrifuge: first for  at  to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for  at maximum speed (> ) to clear the membrane. Discard the flow-through and the collection tube.
Centrifuge: 1000 34
Centrifuge: 12000 34
For optimal results, ensure that the spin column is placed in the centrifuge in the same orientation at each spin step (for example, always with the hinge pointing to the outside of the centrifuge); ensuring the liquid follows the same path through the membrane for binding and elution can slightly improve yield and consistency.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer column to a new collection tube and add  gDNA Wash Buffer.
500 µl
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and invert a few times, so that the wash buffer reaches the cap. Centrifuge immediately for  at maximum speed (), and discard the flow through. The collection tube can be tapped on a paper towel to remove any residual buffer before reusing it in the next step.
Centrifuge: 12000 34
Inverting the spin column containing wash buffer prevents salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Reinsert the column into the collection tube. Add  gDNA Wash Buffer and close the cap. Centrifuge immediately for  at maximum speed (> ), then discard the collection tube and flow through.
500 µl
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included).
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  –  preheated () gDNA Elution Buffer, close the cap and incubate at  for .
35 µl
100 µl
60 °C
60 Room temperature
Elution in 100 μl is recommended, but smaller volumes can be used and will result in more concentrated DNA but a reduced yield (20 – 25 % reduction when using 35 μl). Eluting with preheated elution buffer will increase yields by ~ 20 – 40 % and eliminates the need for a second elution. For applications in which a high DNA concentration is required, using a small elution volume and then eluting again with the eluate may increase yield (~ 10 %). The elution buffer (10 mM Tris-Cl, pH 9.0, 0.1 mM EDTA) offers strong protection against enzymatic degradation and is optimal for long term storage of DNA. However, other low-salt buffers or nuclease-free water can be used if preferred. For more details on optimizing elution, please refer to “Considerations for Elution & Storage” in the product manual.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Centrifuge for  at maximum speed (> ) to elute the gDNA.
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for   – .
400 µl
Thorough mixing is essential for optimal results.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer the lysate/binding buffer mix (~ ) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Proceed immediately to step 8.
600 µl
Avoid touching the upper column area with lysate/binding mix and avoid transferring foam that may have formed during lysis. Any material that touches the upper area of the column, including any foam, may lead to salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and centrifuge: first for  at  to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for  at maximum speed (> ) to clear the membrane. Discard the flow-through and the collection tube.
Centrifuge: 1000 34
Centrifuge: 12000 34
For optimal results, ensure that the spin column is placed in the centrifuge in the same orientation at each spin step (for example, always with the hinge pointing to the outside of the centrifuge); ensuring the liquid follows the same path through the membrane for binding and elution can slightly improve yield and consistency.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer column to a new collection tube and add  gDNA Wash Buffer.
500 µl
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and invert a few times, so that the wash buffer reaches the cap. Centrifuge immediately for  at maximum speed (), and discard the flow through. The collection tube can be tapped on a paper towel to remove any residual buffer before reusing it in the next step.
Centrifuge: 12000 34
Inverting the spin column containing wash buffer prevents salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Reinsert the column into the collection tube. Add  gDNA Wash Buffer and close the cap. Centrifuge immediately for  at maximum speed (> ), then discard the collection tube and flow through.
500 µl
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included).
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  –  preheated () gDNA Elution Buffer, close the cap and incubate at  for .
35 µl
100 µl
60 °C
60 Room temperature
Elution in 100 μl is recommended, but smaller volumes can be used and will result in more concentrated DNA but a reduced yield (20 – 25 % reduction when using 35 μl). Eluting with preheated elution buffer will increase yields by ~ 20 – 40 % and eliminates the need for a second elution. For applications in which a high DNA concentration is required, using a small elution volume and then eluting again with the eluate may increase yield (~ 10 %). The elution buffer (10 mM Tris-Cl, pH 9.0, 0.1 mM EDTA) offers strong protection against enzymatic degradation and is optimal for long term storage of DNA. However, other low-salt buffers or nuclease-free water can be used if preferred. For more details on optimizing elution, please refer to “Considerations for Elution & Storage” in the product manual.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Centrifuge for  at maximum speed (> ) to elute the gDNA.
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for   – .
400 µl
Thorough mixing is essential for optimal results.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer the lysate/binding buffer mix (~ ) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Proceed immediately to step 8.
600 µl
Avoid touching the upper column area with lysate/binding mix and avoid transferring foam that may have formed during lysis. Any material that touches the upper area of the column, including any foam, may lead to salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and centrifuge: first for  at  to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for  at maximum speed (> ) to clear the membrane. Discard the flow-through and the collection tube.
Centrifuge: 1000 34
Centrifuge: 12000 34
For optimal results, ensure that the spin column is placed in the centrifuge in the same orientation at each spin step (for example, always with the hinge pointing to the outside of the centrifuge); ensuring the liquid follows the same path through the membrane for binding and elution can slightly improve yield and consistency.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Transfer column to a new collection tube and add  gDNA Wash Buffer.
500 µl
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Close the cap and invert a few times, so that the wash buffer reaches the cap. Centrifuge immediately for  at maximum speed (), and discard the flow through. The collection tube can be tapped on a paper towel to remove any residual buffer before reusing it in the next step.
Centrifuge: 12000 34
Inverting the spin column containing wash buffer prevents salt contamination in the eluate.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Reinsert the column into the collection tube. Add  gDNA Wash Buffer and close the cap. Centrifuge immediately for  at maximum speed (> ), then discard the collection tube and flow through.
500 µl
Centrifuge: 12000 34
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included).
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Add  –  preheated () gDNA Elution Buffer, close the cap and incubate at  for .
35 µl
100 µl
60 °C
60 Room temperature
Elution in 100 μl is recommended, but smaller volumes can be used and will result in more concentrated DNA but a reduced yield (20 – 25 % reduction when using 35 μl). Eluting with preheated elution buffer will increase yields by ~ 20 – 40 % and eliminates the need for a second elution. For applications in which a high DNA concentration is required, using a small elution volume and then eluting again with the eluate may increase yield (~ 10 %). The elution buffer (10 mM Tris-Cl, pH 9.0, 0.1 mM EDTA) offers strong protection against enzymatic degradation and is optimal for long term storage of DNA. However, other low-salt buffers or nuclease-free water can be used if preferred. For more details on optimizing elution, please refer to “Considerations for Elution & Storage” in the product manual.
?. [PART 2: GENOMIC DNA BINDING AND ELUTION]
Centrifuge for  at maximum speed (> ) to elute the gDNA.
Centrifuge: 12000 34
?. [PART 1: SAMPLE LYSIS]
Weigh the appropriate tissue amount and place in a 1.5 ml microfuge tube (see table below or Choosing Input Amounts for recommended input amounts).  AB1STARTING MATERIALRECOMMENDED INPUT AMOUNT2Rodent tailup to 25 mg3Brainup to 12 mg4Fibrous tissue (muscle, heart)up to 25 mg5Ear clips, skinup to 10 mg6Liver, lungup to 15 mg7Spleen, kidnecup to 10 mg
AB1STARTING MATERIALRECOMMENDED INPUT AMOUNT2Rodent tailup to 25 mg3Brainup to 12 mg4Fibrous tissue (muscle, heart)up to 25 mg5Ear clips, skinup to 10 mg6Liver, lungup to 15 mg7Spleen, kidnecup to 10 mg