Feb 12, 2024

DNA isolation from cattle tissues: blood, semen or any kind of tissues, including ear biopsies

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3xojeg25/v1
DNA isolation from cattle tissues: blood, semen or any kind of tissues, including ear biopsies
  • Cecile CG Grohs1
  • 1Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350, Jouy-en-Josas, France
Cecile Grohs
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3xojeg25/v1
Protocol Citation: Cecile CG Grohs 2024. DNA isolation from cattle tissues: blood, semen or any kind of tissues, including ear biopsies. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xojeg25/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: January 30, 2024
Last Modified: February 22, 2024
Protocol  Integer ID: 94392
Keywords: dna isolation from cattle tissue, isolate dna, routine method for isolate dna, dna isolation, cattle tissue, available frozen semen straw, dna, ear biopsy, genomic dna, including ear biopsy, removal of protein, precipitation of genomic dna, biopsy, washing of sample, semen, other tissue, tissue, different kinds of tissue
Abstract
Here we describe a routine method for isolate DNA from different kinds of tissues: commercially available frozen semen straws, blood, ear biopsies, or other tissues.
This protocol is based on a salting-out method and uses several commercially available solutions.

It consists of several steps: washing of samples, lysis, removal of proteins and precipitation of genomic DNA. This protocol was used to isolate hundredsod samples for years.
Guidelines
For recovering DNA from blood, use K3-EDTA tubes.
Salting out is a good method to obtain DNA, as it avoids using phenol/chloroform and allows to recover DNA from different qualities of blood.
Materials
Isopropanol
70% ethanol
Puregene Tissue Kit QiagenCatalog #158063
Puregene blood kitQiagenCatalog #158106
Proteinase K, 2mLQiagenCatalog #19131
Tris(2-carboxyethyl)phosphine hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #646547-10X1ML Buffer RLTQiagenCatalog #79216
1X PBS (Phosphate-buffered saline )
DNA LoBind Tubes 2.0 mLEppendorfCatalog #30108078
2mL tubes
2 X50mL tubes for each blood sample
Centrifuge for 2mL and 50mL tubes, blood tubes
3D heating rocker

Safety warnings
See Safety Data Sheets for warnings and safety hazards.
Before start
As we use commercial sperm straws to perform our extractions, we do not always know the composition of these straws, the quantity of material contained, the nature of the diluents and preservatives used. This is why it is sometimes necessary to use several straws to obtain enough material for sequencing. It is also sometimes wise to perform several washes (see step 3) to eliminate contaminants from diluents and preservatives.
Prepare reagents
10m

For semen straws, Immidiately before use, prepare a mix containing RLT buffer (Qiagen) and TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] to a final volume of 500µL per sample as follow:
-450 µL RLT
-50 µL TCEP

Prepare samples
10m
Use DNA LoBind tubes at this stage (strongly recommended for sperm, not mandatory for other tissues).
For semen:
  • Empty the 200 µL Straw in a 2 mL tube by cutting the two ends of the straw
  • Rince the straw with 200 µL 1X PBS at Room temperature

For ear biopsy:
  • Open the device
  • Transfer the biopsy in a 2 mL tube
  • Remove extra preservative liquid and the plastic ball

For blood:
  • Pour the 5 mL blood in a 50 mL tube

For other tissues:
  • Cut a 150 mg piece and put it in a 2 mL tube

Wash step

For semen:
  • Add 800 µL more PBS (up to 1 mL 1X PBS )
  • Pellet 1000 x g at Room temperature during 00:05:00
  • Discard the supernatant

Second wash is optional (no significant impact observed)
  • Re-suspend in 1 mL 1X PBS
  • Pellet 1000 x g at Room temperature during 00:05:00
  • Discard the supernatant

For blood:
  • Add 15 mL (i.e. three times the initial volume) RBC reagent from Puregene blood kit
  • Shake vigorously and incubate 00:05:00 at Room temperature
  • Pellet white blood cells 3000 x g, 10°C, 00:15:00
30m
Lysis
3h 30m
Continue with Qiagen Puregene kit adapted as follow

Step one
For semen:
  • Add 500 µL of RLT/TCEP mix
  • Vortex by pulsing at max speed
  • Incubate 00:05:00 on ice
  • Add 500 µL CLS

For tissues, including ear biopsies:
  • Add 800 µL CLS
  • Add 60 µL Proteinase K

For blood:
  • Add 5 mL CLS (i.e. the initial blood volume)
5m
Step 2
  • Mix by inversion (about 25 inversions)
  • Incubate from01:00:00 to 03:00:00 , depending on the lysis process, 55 °C
  • on a rotating shaker at 200 rpm

Check that no undigested parts remain after lysis
4h
Protein precipitation
30m 30s
For semen and tissues:
  • Add 200 µL of Protein Precipitation Solution
  • Vortex 00:00:15 and incubate 00:05:00 on ice
  • Pellet 16000 x g, 4°C, 00:05:00

For blood:
  • Add 1.65 mL of Protein Precipitation Solution (for 5mL blood)
  • Vortex 00:00:15 and incubate 00:05:00 on ice
  • Pellet 3000 x g, 10°C, 00:15:00

30m 30s
DNA precipitation
  • Transfert the supernatant to a new tube containing
600 µL of Isopropanol (semen) - 2 mL tube
800 µL of Isopropanol (tissues) - 2 mL tube
5 mL of Isopropanol (blood) - 50 mL tube
  • Carrefully invert the tube 25-50X times to form the pellet
  • Incubate 00:05:00 Room temperature

For semen and tissues:
  • Centrifuge as previously
  • Discard supernatant

For blood:
  • Pick up the pellet with a lab pipet and transfer it into a 2 mL tube
  • Discard supernatant left

5m
Wash DNA
20m
  • Add 600 µL 70% Ethanol
  • Centrifuge16000 rpm, 4°C, 00:05:00
  • Discard supernatant
  • Allow the ethanol to evaporate, without drying the pellet 00:15:00

20m
DNA resuspension
20m
  • Add TE or EB buffer, depending on the subsequent analysis and usual procedure
We recommend 50 µL for semen or tissue pellets, and 100 µL to 150 µL for pellets from 5mL blood
  • Do not hesitate to heat for 01:00:00 at 50 °C on gentle agitation 100 rpm
1h
DNA quantity control
  • Measure the O.D. with a Nanodrop® device to obtain the DNA concentration
  • Dilute with extra TE/EB if you choose to standardize concentrations
  • Measure until the concentration is as expected

DNA quality control
  • Load the DNA on a 1% agarose gel in 0.5X TBE with Ethidium Bromide
  • Perform an electrophoresis 01:00:00 at 70 V
1h
Protocol references
See protocole for DNA isolation from cattle semen for long read sequencing at DOI dx.doi.org/10.17504/protocols.io.j8nlkw1qwl5r/v1