Genomic DNA extraction from environmental samples having low microbial biomass and different characteristics
Creators: name: Singh, Abhijeet
Publisher: name: Zenodo
Year: 2025
Subjects: DNA extraction

description: * Correspondence: Abhijeet Singh (abhijeet.singh@geo.uu.se)

Description

This protocol is aimed to extract the genomic DNA from less valuable samples* having low microbial biomass and different physical characteristics in a single combined extraction round. Also, the protocol focuses on the extraction of DNA from microbial community and is particularly not concerned with the microbial biomass quantification per unit sample weight. However, quantification per unit weight of sample can be done if all the samples are weighted during preparation step. This protocol consists of two main steps of sample preparation and DNA extraction. Although this protocol is based on the DNA extraction using Qiagen DNeasy® PowerSoil® Pro Kit, yet the protocol can be used for the DNA extraction with any DNA isolation kit.

* Samples not of the archaeological or forensic importance and has ample amounts

 

Pre-preparation

¤ Proper risk assessment and toxicity examinations of samples, chemicals used and kit components should be done

¤ Phenol:Chloroform:Isoamyl Alcohol and Guanidine thiocyanate are toxic chemicals and care should be taken while working with them

¤ Working in ventilated chemical hood is highly recommended

 

Sample preparation

¤ Add basalt and straw samples to respective tube up to half the volume of 50 ml centrifuge tube

               #  Individual samples can be weighted for DNA quantification per unit sample weight

¤ Add Tris-HCl buffer with pH 7.5 up to the 40 ml volume of the centrifuge tube

               #  The pH and volume of buffer can be adjusted accordingly based on the pH and water absorption properties of sample 

¤ Mounted the samples on a laboratory orbital shaker in horizontal position and shake for 5 hr at 80 rmp

               #  Samples with less abrasive properties like straw can be shaken at higher frequency and samples like sand, rock etc. which has high abrasive properties should                      be shaken at lower frequencies

¤ Collect the liquid fraction in a new 50 ml centrifuge tube

               #  The liquid samples should have the suspended finer particles. In case of low recovery of liquid and low suspended material, the shaking step can be repeated                     with addition of new Tris-HCl buffer

               # Centrifuge tube of 15 ml can also be used in case 50 ml centrifuge is not available

¤ Centrifuge for 10 min at 4000 g

               #  Adjust the time and centrifuge speed according to sample characteristics

¤ Wash the pellet twice with Tris-HCl buffer with pH 7.5 and centrifugation for 10 min at 4000 g

               #  To remove the excess salts and other soluble chemical species. In case the sample has high amount of humic acid and other organic acids, the samples can be                    washed with 5.5 M Guanidine thiocyanate solution

DNA extraction

·                Briefly spin the PowerBead Pro lysis tube to concentrate lysing bead matrix at the bottom of tube

·                Add approximately 200-250 mg of the sample pellet to lysis tube. Use replicate tubes according to the amount of pellet

·                Add 600 µl of solution CD1 to the lysis tube. The required volume of CD1 (volume per sample times the number of replicates) can also be used to wash the                  remnant of pellet in centrifuge tube and transferred to lysis tube

·                Carefully add 200 µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) to lysis tube

·                Lyse the samples at maximum frequency for 1 min for instrument similar to FastPrep-24™ homogenizer, for 5-6 min for instrument similar to TissueLyser and for                  5-10 min for instrument similar to VortexGenie

·                Centrifuge the lysis tube for 1 min at >15000 g

·                Transfer the liquid fraction to a new 2 ml centrifuge tube

·                Add equal volume of solution CD3 and mix well for 10 s or vortex for 5 s

·                Load 650 µl volume to MB spin column and centrifuge for 1 min at 15000 g

·                Discard the flow-through in a waste container

·                Repeat the loading and centrifuge step until whole volume of solution CD3 passed through spin filter

·                Place the spin column into a new collection tube

·                Add 500 µl of solution EA to spin column and centrifuge for 1 min at 15000 g

·                Discard the flow-through in a waste container

·                Add 500 µl of solution C5 to spin column and centrifuge for 1 min at 15000 g

·                Discard the flow-through in a waste container

·                Centrifuge empty spin column for 2 min at >15000 g

·                Place the spin column into a new 1.5 ml elution tube

·                Add 50–100 µl of solution C6 to the centre of the white filter membrane. For concentrated samples use lower volume of solution C6

·                Incubate for 1-2 min followed by centrifuge for 1 min at 15000 g

·                Save the flow-through as it will contain DNA

Post-processing

¤ In case the lids of tubes are broken during centrifuge, transfer DNA to a new tube

¤ Mark the DNA tube with appropriate sample name and date

¤ Maintain proper note in lab-book

¤ For DNA quantification, Qubit or Nanodrop can be used. Quantification using fluorescence methods like Qubit is more accurate

¤ DNA quantification per unit sample weight can now be done based on weight of sample used and the obtained DNA concentration

¤ Prior to sequencing or PCR analysis, samples can be normalized for DNA concentration per unit volume, especially according to the sample with lowest DNA    concentration