TITLE: DNA isolation from cattle tissues: blood, semen or any kind of tissues, including ear biopsies
AUTHORS: Cecile CG Grohs

[DESCRIPTION]
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.

[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.

[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.

[STEPS]
SECTION: Lysis
3. Continue with Qiagen Puregene kit adapted as follow

Step one
For semen: 
Add 500 µLof RLT/TCEP mix
Vortex by pulsing at max speed
Incubate 5 min 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)
SECTION: Protein precipitation
5. For semen and tissues:
Add 200 µLof Protein Precipitation Solution 
Vortex 15 s and incubate 5 min on ice
Pellet 16000 x g, 5 min, 4 °C

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

SECTION: DNA precipitation
6. 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 5 min  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

SECTION: Wash DNA
7. Add 600 µL 70% Ethanol
Centrifuge16000 rpm, 5 min, 4 °C
Discard supernatant
Allow the ethanol to evaporate, without drying the pellet 15 min 

SECTION: DNA resuspension
8. 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 60 min at 50 °C on gentle agitation  
SECTION: Prepare samples
2. Use DNA LoBind tubes at this stage (strongly recommended for sperm, not mandatory for other tissues).
SECTION: Prepare reagents
1. 
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  
-50 µL

SECTION: Prepare samples
2.1. 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

SECTION: Prepare samples
2.2. Wash step

For semen:
Add 800 µL more PBS (up to 1 mL 1X PBS  )
Pellet 1000 x g at Room temperature during 5 min
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 5 min
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 5 min at Room temperature 
Pellet white blood cells 3000 x g, 15 min, 10 °C 
SECTION: Lysis
4. Step 2
Mix by inversion (about 25 inversions)
Incubate from60 min  to 180 min, depending on the lysis process, 55 °C 
on a rotating shaker at  

Check that no undigested parts remain after lysis
SECTION: DNA resuspension
9.  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 60 min at 70 V