Sep 16, 2024

Public workspaceEthanol precipitation of small DNA fragments

DOI
dx.doi.org/10.17504/protocols.io.n2bvj7jnvk5w/v1
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Eva Petrova
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.n2bvj7jnvk5w/v1
Protocol Citation: Eva Petrova, Roey Angel 2024. Ethanol precipitation of small DNA fragments. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj7jnvk5w/v1
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: May 24, 2018
Last Modified: September 16, 2024
Protocol Integer ID: 12439
Abstract
This protocol is for a simple ethanol precipitation of small fragments and/or small amounts of DNA. Note that this protocol simply concentrates your sample and removes enough salts/enzymes for ligation to be successful. All DNA fragments from your digest will still be present in your pellet.
See here for a discussion of theory behind ethanol precipitation of DNA fragments.
Guidelines
3M Sodium Acetate, 0.11M MgCl2 pH 5.5
24.6 g sodium acetate
0.952g MgCl2 anhydrous
Dissolve in about 60 ml DDW Adjust pH with glacial acetic acid (>8 ml), Dilute to 100 ml with DDW and filter sterilize.
Materials
MATERIALS
ReagentPellet Paint® NF Co-PrecipitantSigma AldrichCatalog #70748-3
Protocol materials
ReagentPellet Paint® NF Co-PrecipitantMerck MilliporeSigma (Sigma-Aldrich)Catalog #70748-3
Materials, Step 3
Before start
  • Absolute Ethanol (100% = 200 proof) at -80o C
  • 70% ethanol at -80o C (don’t cool ethanol for too long or water will freeze)
  • Tabletop centrifuge
  • -80°C freezer
Transfer the sample to 1.5 mL eppendorf tube.
Adjust the salt conc. of the sample to 0.3M sodium acetate and 0.01M MgCl2.
Concentration0.3 Molarity (M) sodium acetate
Concentration0.01 Molarity (M) MgCl2
Add 1 µl of Pellet Paint NF.
Amount1 µL Pellet Paint NF
ReagentPellet Paint® NF Co-PrecipitantSigma AldrichCatalog #70748-3
Add 1 µl of non labeled genomic DNA e.g. from E.coli (this acts as a carrier and increases precipitation yield).
Amount1 µL genomic DNA (E.coli)
Add 2 volumes cold absolute ethanol to sample.
Incubate 2-3 hr at -80°C.  The long incubation time is critical for small fragments.
Temperature-80 °C
Duration02:00:00
Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
Command
max. speed
CENTRIFUGE
Temperature0 °C
Duration00:30:00
Remove supernatant.
Wash with 750-1000 μL cold 70% ethanol.  Another critical step for small fragments under 200 base pairs. Washing involves adding the ethanol and inverting several times.
Amount750 µL cold 70% ethanol
Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
Command
max. speed
CENTRIFUGE
Temperature0 °C
Duration00:30:00
Repeat washing procedure and centrifuge again.
Go togo to step #9
Let air dry on benchtop.
Resuspend in an appropriate volume of H2O.