Feb 11, 2020
DNA extraction from whole blood using simple salting out procedure
- Schuele Lab1,
- Faria Zafar1
- 1Department Pathology, Stanford University School of Medicine
Protocol Citation: Schuele Lab, Faria Zafar 2020. DNA extraction from whole blood using simple salting out procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.bbzqip5w
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 in our group and it is working.
Created: January 31, 2020
Last Modified: February 11, 2020
Protocol Integer ID: 32528
Keywords: DNA, DNA extraction, blood, whole blood, human blood, salting out, nuclei lysis,
Abstract
The purpose of this protocol is high-molecular weight DNA extraction from whole blood for genetic analyses, including no amplification long-range sequencing.
CITATION
Attachments
Guidelines
Storage Vial labeling:
3 lines, 6pt bold font; 1.5” label
a) Subject ID#: four digit SUSL ID (e.g. SUSL-2345), Sample type: WBC
b) Date of blood draw: MM/DD/YY
c) Study: [Name Study], Initials
Materials
MATERIALS
Sodium chlorideSigma AldrichCatalog #S3014
Ammonium chloride ( ≥ 99.5 %)Sigma AldrichCatalog #A9434
Potassium bicarbonate (≥ 99.5 %)Sigma AldrichCatalog #90339
EDTA 500 mM Solution pH 8.0 ULTROL® GradeMerck MilliporeCatalog #324504
Trizma® hydrochloride / Tris-HClMerck Millipore SigmaCatalog #T5941
Ethyl Alcohol 200 Proof (GR ACS)Millipore SigmaCatalog #EX0276
2-Propanol (99.5 %)Millipore SigmaCatalog #278475
Proteinase K from Tritirachium albumMillipore SigmaCatalog #P2308
Note
If using different vendors, choose molecular biology grade reagents.
Buffers and Solution Recipes
10X Red blood cell (RBC) lysis buffer (1l)
| NH4Cl | 82.91 g | |
| KHCO3 | 10.01 g | |
| EDTA 0.5 M | 20 ml | |
| complete to 1000 ml with autoclaved dH2O | ||
* dilute to 1x before use with autoclaved dH2O
1 M Tris-HCl, pH 8 (used to make nucleolysis buffer and TE)
| Tris -HCl | 15.7 g | |
| Bring volume to ~ 95ml with autoclaved ddH2O | ||
| measure & adjust pH to 8 | ||
| Bring final volume up to 100 ml with autoclaved ddH2O | ||
10X Nucleus lysis buffer (100ml)
| NaCl | 23.38 g | |
| Tris-HCl (1 M, pH = 8) | 10 ml | |
| EDTA 0.5 M | 4 ml | |
| complete to 100 ml with autoclaved ddH2O | ||
* dilute to 1x before use with autoclaved ddH2O
20 % SDS (used end of Day 1 for DNA extraction to poke holes in cell membrane)
| SDS | 20 g | |
| autoclaved ddH2O | 80 ml |
5 M NaCl
| NaCl | 146.1 g | |
| Bring volume to 500 ml with autoclaved dH2O | ||
70 % EtOH (used Day 2 of DNA extraction to “clean” DNA)
| 100 % EtOH | 35 ml | |
| ddH2O | 15 ml |
TE (used at end of DNA extraction to re-suspend DNA and prepare for storage)
| 1 M Tris-HCl | 1 ml | |
| 0.5 M EDTA | 0.2 ml | |
| Bring volume to 100 ml with autoclaved ddH2O | ||
Proteinase K reconstitution:
Solutions can be prepared in 25 millimolar (mM) Tris-HCl buffer , 8.0 , containing 1 millimolar (mM) Calcium chloride . Amounts for typical usage is 50 μg/ml – 200 μg/ml . Solutions are stable at 8 at 4 °C . It is stable in a broad range of environments: pH, buffer salts, detergents (SDS), and temperature. In the presence of 0.1 – 0.5 % SDS, proteinase K retains activity and will digest a variety of proteins and nucleases in DNA preparations without compromising the integrity of the isolated DNA.
Materials
- 10 ml EDTA collection tube (K2EDTA, 5.4mg; 16 x 100mm; 10mL; Pink)
- 1000 µl filter pipette tips
- Gloves
- Bleach (e.g. Clorox, as 20 % fresh solution)
Equipment
- Centrifuge: Sorvall Legend XTR, or SorvallT 6000B, H10000B
- Centrifuge: Eppendorf 5417R
- Rotator
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Red blood cell lysis
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'.
In the morning take out tubes of blood from the -80 °C freezer. Record ID numbers and put in fridge (4 °C ) to thaw until later in the afternoon.
FROZEN blood37 steps
Once blood has thawed, invert several times or place on rotator for a couple of minutes.
Decant the blood sample into 50 ml conical tube.
Rinse blood tube with 1 mL of 1x RBC lysis buffer and add to 50ml conincal tube.
Add 1x RBC lysis buffer up to 50 mL .
Place on shaker at Room temperature for 00:30:00 .
Note
Note: Blood solution becomes transparent.
Centrifuge 00:15:00 at 2000 rpm at Room temperature .
Discard supernatant in container with 20 % bleach .
Note
Note: The pellet contains the nuclei.
Note
If direct processing with Nuclei lysis, day 1, then continue after this step with Nuclei lysis (step 12).
Add 700 µL 1x RBC lysis buffer 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).
Transfer to 1.5 ml pre-labeled clear Eppendorf tube.
Centrifuge 00:05:00 at 2000 rpm at Room temperature .
Using a 1000 µl pipette tip, discard the supernatant in container with 20 % bleach .
Pellets can be frozen at this point at -80 °C .
Nuclei lysis (Day 1, afternoon)
Nuclei lysis (Day 1, afternoon)
Add 3 mL Nucleus Lysis Buffer and close tube tightly.
Pipette up and down to resuspend pellet.
Add 300 µL of 10 % SDS and 70 µL Proteinase K (10 mg/ml ).
Mix by gently swirling tube.
Incubate Overnight in water bath at 55 °C .
DNA extraction (Day 2, morning)
DNA extraction (Day 2, morning)
Add 1 mL of 5 Molarity (M) NaCl to the tubes.
Close caps tightly and shake vigorously.
Note
Note: Very important to shake thoroughly at this step to precipitate proteins.
Centrifuge for 00:20:00 at 3000 rpm at Room temperature .
While centrifuging, label 2x 15ml conical tubes per sample.
Transfer supernatant to a 15 ml conical tube and then repeat centrifugation for 00:20:00 at 3000 rpm
Note
Note: The supernatant contains the DNA.
Transfer supernatant to next 15 ml conical tube.
Add equal volume of ice-cold isopropanol (usually about 4 mL ).
Invert tubes several times and DNA will precipitate.
Note
Note: You should be able to see white strings (this is the DNA!).
Centrifuge for 00:05:00 at 3000 rpm .
Discard supernatant.
Add 1 mL 70 % ethanol to each tube.
Centrifuge for 00:05:00 at 3000 rpm .
Carefully pipette off ethanol to not dislodge the DNA pellet.
Leave DNA to dry in uncapped tubes overnight.
Dissolve DNA
Dissolve DNA
Add 250 µL TE and allow DNA to dissolve in 15 ml conical tube stored in fridge (4 °C ).
Once completely dissolved, transfer to labeled sterile Eppendorf tube.
Label top with subject ID. Label side with date of extraction and concentration.
Spec all samples using Nanodrop. Dilute samples to stock at 250 ng/μl – 350 ng/μl and to working dilution at 10 ng/μl .
Transfer samples into barcode tubes and log into DNA bank.
Citations
S. A. Miller, D. D. Dykes, H. F. Polesky. A simple salting out procedure for extracting DNA from human nucleated cells
10.1093/nar/16.3.1215