TITLE: DNA extraction from whole blood using simple salting out procedure
AUTHORS: Birgitt Schuele, Faria Zafar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is high-molecular weight DNA extraction from whole blood for genetic analyses, including no amplification long-range sequencing.</div></div>

[STEPS]
?. [Red blood cell lysis]
If you use FROZEN blood, start with step-case 'FROZEN blood'. If FRESH blood is used, select step-case 'FRESH blood'.
?. [Red blood cell lysis]
Decant the blood sample into 50 ml conical tube.
?. [Red blood cell lysis]
Add 1x RBC lysis buffer up to .
50 ml
?. [Red blood cell lysis]
Place on shaker at  for .
4 Room temperature
Note: Blood solution becomes transparent.
?. [Red blood cell lysis]
Centrifuge  at  at .
Centrifuge: 2000 33
4 Room temperature
?. [Red blood cell lysis]
Discard supernatant in container with .
[bleach]
Note: The pellet contains the nuclei.
If you continue with processing "Nuclei lysis, day 1", then continue after this step with Nuclei lysis (step 13) and leave pellet in 50ml conical tube.
?. [Red blood cell lysis]
Add  and close the tube tightly. Pipette up and down to resuspend the pellet (cleans up the nuclei pellet from additional RBCs and allows for storage in small tubes).
[1x RBC lysis buffer]
?. [Red blood cell lysis]
Transfer to 1.5 ml pre-labeled clear Eppendorf tube.
?. [Red blood cell lysis]
Centrifuge  at  at .
Centrifuge: 2000 33
4 Room temperature
?. [Red blood cell lysis]
Using a 1000 µl pipette tip, discard the supernatant in container with .
[bleach]
?. [Red blood cell lysis]
Pellets can be frozen at this point at .
-80 °C
?. [Nuclei lysis (Day 1, afternoon)]
Add  and close tube tightly.
[Nucleus Lysis Buffer]
?. [Nuclei lysis (Day 1, afternoon)]
Pipette up and down to resuspend pellet.
?. [Nuclei lysis (Day 1, afternoon)]
Add  of  and  ( stock solution).
300 µl
[SDS]
[Proteinase K]
The total volume is about 3370 plus the volume of the pellet for the nuclei lysis.If 70ul of 10mg/ml proteinase K is added, it results in  a dilution of ~1:50, which will be a final proteinase K concentration of 200ug/ml.
?. [Nuclei lysis (Day 1, afternoon)]
Mix by gently swirling tube.
?. [Nuclei lysis (Day 1, afternoon)]
Incubate  in water bath at .
55 °C
Temperature is at 55C as proteinase K is resistant to denaturation by heat and will continue to function as long as the temperature/concentration is not too high, wherease most mucleases are denatureated at this temparature.
?. [DNA extraction (Day 2, morning)]
Add  of  to the tubes.
1 ml
[NaCl]
?. [DNA extraction (Day 2, morning)]
Close caps tightly and shake vigorously.
Note: Very important to shake thoroughly at this step to precipitate proteins.
?. [DNA extraction (Day 2, morning)]
Centrifuge for  at  at .
Centrifuge: 3000 33
-80 Room temperature
?. [DNA extraction (Day 2, morning)]
While centrifuging, label 2x 15ml conical tubes per sample.
?. [DNA extraction (Day 2, morning)]
Transfer supernatant to a 15 ml conical tube and then repeat centrifugation for  at
Centrifuge: 3000 33
Note: The supernatant contains the DNA.
?. [DNA extraction (Day 2, morning)]
Transfer supernatant to next 15 ml conical tube.
?. [DNA extraction (Day 2, morning)]
Add equal volume of ice-cold isopropanol (usually about ).
4 ml
?. [DNA extraction (Day 2, morning)]
Invert tubes several times and DNA will precipitate.
Note: You should be able to see white strings (this is the DNA!).
?. [DNA extraction (Day 2, morning)]
Centrifuge for  at .
Centrifuge: 3000 33
?. [DNA extraction (Day 2, morning)]
Discard supernatant.
?. [DNA extraction (Day 2, morning)]
Add  to each tube.
1 ml
[ethanol]
?. [DNA extraction (Day 2, morning)]
Centrifuge for  at .
Centrifuge: 3000 33
?. [DNA extraction (Day 2, morning)]
Carefully pipette off ethanol to not dislodge the DNA pellet.
?. [DNA extraction (Day 2, morning)]
Leave DNA to dry in uncapped tubes overnight.
?. [Dissolve DNA]
Add  to  and allow DNA to dissolve in 15 ml conical tube stored in fridge ().
100 µl
[TE]
4 °C
?. [Dissolve DNA]
Once completely dissolved, transfer to labeled sterile Eppendorf tube.
?. [Dissolve DNA]
Label top with subject ID. Label side with date of extraction and concentration.
?. [Dissolve DNA]
Spec all samples using Nanodrop. Dilute samples to stock at  –  and to working dilution at .
?. [Dissolve DNA]
Transfer samples into barcode tubes and log into DNA bank.
?. [Red blood cell lysis]
In the morning take out tubes of blood from the  freezer. Record ID numbers and put in fridge () to thaw until later in the afternoon.
-80 °C
4 °C
?. [Red blood cell lysis]
Once blood has thawed, invert several times or place on rotator for a couple of minutes.
?. [Red blood cell lysis]
Decant the blood sample into 50 ml conical tube.
?. [Red blood cell lysis]
Add 1x RBC lysis buffer up to .
50 ml
?. [Red blood cell lysis]
Place on shaker at  for .
4 Room temperature
Note: Blood solution becomes transparent.
?. [Red blood cell lysis]
Centrifuge  at  at .
Centrifuge: 2000 33
4 Room temperature
?. [Red blood cell lysis]
Discard supernatant in container with .
[bleach]
Note: The pellet contains the nuclei.
If direct processing with Nuclei lysis, day 1, then continue after this step with Nuclei lysis (step 12).
?. [Red blood cell lysis]
Add  and close the tube tightly. Pipette up and down to resuspend the pellet (cleans up the nuclei pellet from additional RBCs and allows for storage in small tubes).
[1x RBC lysis buffer]
?. [Red blood cell lysis]
Transfer to 1.5 ml pre-labeled clear Eppendorf tube.
?. [Red blood cell lysis]
Centrifuge  at  at .
Centrifuge: 2000 33
4 Room temperature
?. [Red blood cell lysis]
Using a 1000 µl pipette tip, discard the supernatant in container with .
[bleach]
?. [Red blood cell lysis]
Pellets can be frozen at this point at .
-80 °C
?. [Nuclei lysis (Day 1, afternoon)]
Add  and close tube tightly.
[Nucleus Lysis Buffer]
?. [Nuclei lysis (Day 1, afternoon)]
Pipette up and down to resuspend pellet.
?. [Nuclei lysis (Day 1, afternoon)]
Add  of  and  ().
300 µl
[SDS]
[Proteinase K]
?. [Nuclei lysis (Day 1, afternoon)]
Mix by gently swirling tube.
?. [Nuclei lysis (Day 1, afternoon)]
Incubate  in water bath at .
55 °C
?. [DNA extraction (Day 2, morning)]
Add  of  to the tubes.
1 ml
[NaCl]
?. [DNA extraction (Day 2, morning)]
Close caps tightly and shake vigorously.
Note: Very important to shake thoroughly at this step to precipitate proteins.
?. [DNA extraction (Day 2, morning)]
Centrifuge for  at  at .
Centrifuge: 3000 33
-80 Room temperature
?. [DNA extraction (Day 2, morning)]
While centrifuging, label 2x 15ml conical tubes per sample.
?. [DNA extraction (Day 2, morning)]
Transfer supernatant to a 15 ml conical tube and then repeat centrifugation for  at
Centrifuge: 3000 33
Note: The supernatant contains the DNA.
?. [DNA extraction (Day 2, morning)]
Transfer supernatant to next 15 ml conical tube.
?. [DNA extraction (Day 2, morning)]
Add equal volume of ice-cold isopropanol (usually about ).
4 ml
?. [DNA extraction (Day 2, morning)]
Invert tubes several times and DNA will precipitate.
Note: You should be able to see white strings (this is the DNA!).
?. [DNA extraction (Day 2, morning)]
Centrifuge for  at .
Centrifuge: 3000 33
?. [DNA extraction (Day 2, morning)]
Discard supernatant.
?. [DNA extraction (Day 2, morning)]
Add  to each tube.
1 ml
[ethanol]
?. [DNA extraction (Day 2, morning)]
Centrifuge for  at .
Centrifuge: 3000 33
?. [DNA extraction (Day 2, morning)]
Carefully pipette off ethanol to not dislodge the DNA pellet.
?. [DNA extraction (Day 2, morning)]
Leave DNA to dry in uncapped tubes overnight.
?. [Dissolve DNA]
Add  and allow DNA to dissolve in 15 ml conical tube stored in fridge ().
[TE]
4 °C
?. [Dissolve DNA]
Once completely dissolved, transfer to labeled sterile Eppendorf tube.
?. [Dissolve DNA]
Label top with subject ID. Label side with date of extraction and concentration.
?. [Dissolve DNA]
Spec all samples using Nanodrop. Dilute samples to stock at  –  and to working dilution at .
?. [Dissolve DNA]
Transfer samples into barcode tubes and log into DNA bank.
?. [Red blood cell lysis]
Rinse blood tube with  of 1x RBC lysis buffer and add to 50ml conincal tube.
1 ml
?. [Red blood cell lysis]
Rinse blood tube with  1xRBC lysis buffer and add to 50ml conical tube.
1 ml