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

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:

-450µL

-50µL

Use DNA LoBind tubes at this stage (strongly recommended for sperm, not mandatory for other tissues).

For semen:

• Empty the 200µL Straw in a 2mL tube by cutting the two ends of the straw
• Rince the straw with 200µL 1X PBS at Room temperature

For ear biopsy:

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

For blood:

• Pour the 5mL blood in a 50mL tube

For other tissues:

• Cut a 150mg piece and put it in a 2mL tube

Wash step

For semen:

• Add 800µL more PBS (up to 1mL 1X PBS )
• Pellet 1000x g,0h 0m 0s at Room temperature during 0h 5m 0s
• Discard the supernatant

Second wash is optional (no significant impact observed)

• Re-suspend in 1mL 1X PBS
• Pellet 1000x g,0h 0m 0s at Room temperature during 0h 5m 0s
• Discard the supernatant

For blood:

• Add 15mL (i.e. three times the initial volume) RBC reagent from Puregene blood kit
• Shake vigorously and incubate 0h 5m 0s at Room temperature
• Pellet white blood cells 3000x g,10°C

Continue with Qiagen Puregene kit adapted as follow

Step one

For semen:

• Add 500µLof RLT/TCEP mix
• Vortex by pulsing at max speed
• Incubate 0h 5m 0s on ice
• Add 500µL CLS

For tissues, including ear biopsies:

• Add 800µL CLS
• Add 60µL Proteinase K

For blood:

• Add 5mL CLS (i.e. the initial blood volume)

Step 2

• Mix by inversion (about 25 inversions)
• Incubate from1h 0m 0s to 3h 0m 0s, depending on the lysis process, 55°C
• on a rotating shaker at 200rpm

Check that no undigested parts remain after lysis

For semen and tissues:

• Add 200µLof Protein Precipitation Solution
• Vortex 0h 0m 15s and incubate 0h 5m 0s on ice
• Pellet 16000x g,4°C

For blood:

• Add 1.65mL of Protein Precipitation Solution (for 5mL blood)
• Vortex 0h 0m 15s and incubate 0h 5m 0s on ice
• Pellet 3000x g,10°C

Transfert the supernatant to a new tube containing 600µL of Isopropanol (semen) - 2mL tube

         `800µL` of Isopropanol (tissues) - `2mL`   tube



         `5mL`    of Isopropanol   (blood)  - `50mL` tube



         

• Carrefully invert the tube 25-50X times to form the pellet
• Incubate 0h 5m 0s 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 2mL tube
• Discard supernatant left

Add 600µL 70% Ethanol* Centrifuge16000rpm,4°C

• Discard supernatant
• Allow the ethanol to evaporate, without drying the pellet 0h 15m 0s

Add TE or EB buffer, depending on the subsequent analysis and usual procedureWe recommend 50µL for semen or tissue pellets, and 100µL to 150µL for pellets from 5mL blood

• Do not hesitate to heat for 1h 0m 0s at 50°C on gentle agitation 100rpm

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 1h 0m 0s at 70 V