Jun 22, 2020
Version 1
Quick Protocol for Extraction and Purification of Genomic DNA V.1
This protocol is a draft, published without a DOI.
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2020. Quick Protocol for Extraction and Purification of Genomic DNA. protocols.io https://protocols.io/view/quick-protocol-for-extraction-and-purification-of-7rwhm7e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2019
Last Modified: March 29, 2021
Protocol Integer ID: 28182
Keywords: gDNA purification, genomic DNA purification, DNA extraction, DNA isolation
Abstract
Genomic DNA Purification Consists of Two Stages:
- Part 1: Sample Lysis
- Part 2: Genomic DNA Binding and Elution
Guidelines
We recommend that first-time users of this kit review the product manual before starting; it provides additional information to consider at various steps. This quick protocol is meant for experienced users. The manual also contains protocols for reaction cleanup and extraction of gDNA from additional sample types.
Materials
MATERIALS
Monarch® Genomic DNA Purification KitNew England BiolabsCatalog #T3010
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
- Store RNase A and Proteinase K at -20 °C.
- Add ethanol (≥ 95 %) to the gDNA Wash Buffer concentrate as indicated on the bottle label.
- Set a thermal mixer (e.g. ThermoMixer®) or, if not available, a heating block to 56 °C for sample lysis.
- Set a heating block to 60 °C. Preheat the appropriate volume of elution buffer to 60 °C (35-100 μl per sample).
Please select the sample material that you want to use:
- Cultured Cells
- Mammalian Whole Blood (non-nucleated)
- Nucleated Whole Blood (birds, reptiles)
- Animal Tissue
Cultured Cells13 steps
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 100 µL cold PBS by pipetting up and down.
Fresh cells: pellet by centrifugation at 1000 x g for 00:01:00 and resuspend in 100 µL 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.
Add 1 µL Proteinase K and 3 µL RNase A to the resuspended pellet and mix by vortexing briefly to ensure the enzymes are efficiently dispersed.
Note
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.
Add 100 µL Cell Lysis Buffer and vortex immediately and thoroughly. The solution will rapidly become viscous.
Incubate for 00:05:00 at 56 °C in a thermal mixer with agitation at full speed (~ 1400 rpm ).
Note
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 2: GENOMIC DNA BINDING AND ELUTION
PART 2: GENOMIC DNA BINDING AND ELUTION
Add 400 µL gDNA Binding Buffer to the sample and mix thoroughly by pulse-vortexing for 00:00:05 – 00:00:10 .
Note
Thorough mixing is essential for optimal results.
Transfer the lysate/binding buffer mix (~ 600 µL ) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Proceed immediately to step 8.
Note
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.
Close the cap and centrifuge: first for 00:03:00 at 1000 x g to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for 00:01:00 at maximum speed (> 12000 x g ) to clear the membrane. Discard the flow-through and the collection tube.
Note
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.
Transfer column to a new collection tube and add 500 µL gDNA Wash Buffer.
Close the cap and invert a few times, so that the wash buffer reaches the cap. Centrifuge immediately for 00:01:00 at maximum speed (12000 x g ), 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.
Note
Inverting the spin column containing wash buffer prevents salt contamination in the eluate.
Reinsert the column into the collection tube. Add 500 µL gDNA Wash Buffer and close the cap. Centrifuge immediately for 00:01:00 at maximum speed (> 12000 x g ), then discard the collection tube and flow through.
Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included).
Add 35 µL – 100 µL preheated (60 °C ) gDNA Elution Buffer, close the cap and incubate at Room temperature for 00:01:00 .
Note
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.
Centrifuge for 00:01:00 at maximum speed (> 12000 x g ) to elute the gDNA.