Feb 12, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.kqdg3xojeg25/v1
  • Cecile CG Grohs1
  • 1Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350, Jouy-en-Josas, France
Cecile Grohs
Open access
DOI: 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
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
ReagentIsopropanolContributed by users
Reagent70% ethanolContributed by users
ReagentPuregene Tissue Kit QiagenCatalog #158063
ReagentPuregene blood kitQiagenCatalog #158106
ReagentProteinase K, 2mLQiagenCatalog #19131
ReagentTris(2-carboxyethyl)phosphine hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #646547-10X1ML ReagentBuffer RLTQiagenCatalog #79216
Reagent1X PBS (Phosphate-buffered saline )
ReagentDNA 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
Attention
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
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:
-Amount450 µL RLT
-Amount50 µL TCEP

Prepare samples
Prepare samples
10m
Use DNA LoBind tubes at this stage (strongly recommended for sperm, not mandatory for other tissues).
For semen:
  • Empty the Amount200 µL Straw in a Amount2 mL tube by cutting the two ends of the straw
  • Rince the straw with Amount200 µL 1X PBS at TemperatureRoom temperature

For ear biopsy:
  • Open the device
  • Transfer the biopsy in a Amount2 mL tube
  • Remove extra preservative liquid and the plastic ball

For blood:
  • Pour the Amount5 mL blood in a Amount50 mL tube

For other tissues:
  • Cut a Amount150 mg piece and put it in a Amount2 mL tube

Wash step

For semen:
  • Add Amount800 µL more PBS (up to Amount1 mL 1X PBS )
  • Pellet Centrifigation1000 x g at TemperatureRoom temperature during Duration00:05:00
  • Discard the supernatant

Second wash is optional (no significant impact observed)
  • Re-suspend in Amount1 mL 1X PBS
  • Pellet Centrifigation1000 x g at TemperatureRoom temperature during Duration00:05:00
  • Discard the supernatant

For blood:
  • Add Amount15 mL (i.e. three times the initial volume) RBC reagent from Puregene blood kit
  • Shake vigorously and incubate Duration00:05:00 at TemperatureRoom temperature
  • Pellet white blood cells Centrifigation3000 x g, 10°C, 00:15:00
30m
Wash
Lysis
Lysis
3h 30m
Continue with Qiagen Puregene kit adapted as follow

Step one
For semen:
  • Add Amount500 µL of RLT/TCEP mix
  • Vortex by pulsing at max speed
  • Incubate Duration00:05:00 on ice
  • Add Amount500 µL CLS

For tissues, including ear biopsies:
  • Add Amount800 µL CLS
  • Add Amount60 µL Proteinase K

For blood:
  • Add Amount5 mL CLS (i.e. the initial blood volume)
5m
Step 2
  • Mix by inversion (about 25 inversions)
  • Incubate fromDuration01:00:00 to Duration03:00:00 , depending on the lysis process, Temperature55 °C
  • on a rotating shaker at Shaker200 rpm

Check that no undigested parts remain after lysis
4h
Incubation
Protein precipitation
Protein precipitation
30m 30s
For semen and tissues:
  • Add Amount200 µL of Protein Precipitation Solution
  • Vortex Duration00:00:15 and incubate Duration00:05:00 on ice
  • Pellet Centrifigation16000 x g, 4°C, 00:05:00

For blood:
  • Add Amount1.65 mL of Protein Precipitation Solution (for 5mL blood)
  • Vortex Duration00:00:15 and incubate Duration00:05:00 on ice
  • Pellet Centrifigation3000 x g, 10°C, 00:15:00

30m 30s
DNA precipitation
DNA precipitation
  • Transfert the supernatant to a new tube containing
Amount600 µL of Isopropanol (semen) - Amount2 mL tube
Amount800 µL of Isopropanol (tissues) - Amount2 mL tube
Amount5 mL of Isopropanol (blood) - Amount50 mL tube
  • Carrefully invert the tube 25-50X times to form the pellet
  • Incubate Duration00:05:00 TemperatureRoom 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 Amount2 mL tube
  • Discard supernatant left

5m
Wash DNA
Wash DNA
20m
  • Add Amount600 µL 70% Ethanol
  • CentrifugeCentrifigation16000 rpm, 4°C, 00:05:00
  • Discard supernatant
  • Allow the ethanol to evaporate, without drying the pellet Duration00:15:00

20m
Wash
DNA resuspension
DNA resuspension
20m
  • Add TE or EB buffer, depending on the subsequent analysis and usual procedure
We recommend Amount50 µL for semen or tissue pellets, and Amount100 µL to Amount150 µL for pellets from 5mL blood
  • Do not hesitate to heat for Duration01:00:00 at Temperature50 °C on gentle agitation Shaker100 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 Duration01:00:00 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