DNA Diagnostic Laboratory - Human and Clinical Genetics (LUMC) - LMDp_Protocols^©

DNA isolation from blood

(last modified December 30, 1999)

Protocol

for the isolation of DNA from leucocytes (kindly provided by Jitske Weegenaar and Els Voorhoeve)

• target group
• responsibilities
• definitions
• description
• durables
• consumables
• chemicals
• solutions
• rules for the DNA-isolation shift
  • safety measures
• DNA-isolation protocol
• references
• trouble shooting

Target group

The technicians who are working in the clinical DNA-diagnostic department.

Responsibilities

The technicians who perform the DNA-isolation are responsible for the blood- and DNA-samples until the samples are handed over to the technicians responsible for further DNA-diagnostic analysis.

Definitions

• (M): available at the storehouse of the Sylvius Laboratory
• RT: room temperature
• HIV: Human Immunodeficiency Virus

Description

In this protocol the isolation of DNA from leucocytes according to the salting out procedure [1] is described. The leucocytes are separated from the red blood cells by differential lysis of the red blood cells, followed by centrifugation to pellet down the intact leucocytes. The supernatant contains the lysed red cells. The DNA containing leucocytes are lysed and the protein debris is removed by the salting out procedure. For dirty samples a phenol-chloroform extraction (ALG009) can be performed. The data related to all samples and other findings/remarks are noted on the form WDNA.

See LP010 for the procedures concerning sample registration, handling, storage, etc.

At the end of this protocol a troubleshooting list is added.

The receipt of all the samples and their processing takes place in the pre-PCR lab. Storage of the DNA-samples is in the cold room. DNA-samples should never enter post-PCR rooms.

Durables

• centrifuge, Varifuge GL or 3.0, Heraeus, Dijkstra Vereenigde
• Denley Store trays 128 * 307
• Denley Store holders 315 * 135 * 277
• dispenser Fortuna Optifix, 1-5 ml, 101-080-33, Merck Vel
• dispenser Fortuna Optifix, 6-30 ml, 101-081-44, Merck Vel
• incubator, model B6, Heraeus, Merck Vel
• label printer, Brady LS 2000, Salm & Kipp
• laminar downflow safetyhood, CA/REV4, Clean Air
• micro centrifuge, 5402, Eppendorf, Merck Vel
• pipetman, micropipetten P20, P200 en P1000, Gilson (M)
• roller bench, Mixer 820, Swelab Instrument
• top-over, Reax 2, Heidolph

NOTE: equivalent durables may be used

Consumables

• blue cap tubes, 50 ml, Greiner (M)
• blue tip, Greiner (M)
• cryotubes 1.0 ml, art.nr. 3-75353k, Nunc, Life Technologies
• flask (hpe), art. nr. 11608, Emergo
• labels #311, Brady, Salm & Kipp
• Pasteur Capillary Pipettes, short size (150 mm), WU Mainz (M)
• Pasteur Pipettes plastic, 3 ml, Greiner (M)
• Safe-Lock tubes, 1.5 ml, Eppendorf (M)
• screw caps (hpe), art. nr. 11567, Emergo
• yellow tips, Greiner (M)

NOTE: equivalent consumables may be used

Chemicals

• acetic acid; HAc 99-100 % p.a. (M)
• ammonium chloride; NH₄Cl p.a. (M)
• EDTA disodium salt p.a. (M)
• ethanol; absolute p.a. (M)
• hydrochloric acid; HCl 36-38% p.a. (M)
• potassium hydrogen carbonate; KHCO₃ p.a. (M)
• pronase; 165921, Boehringer Mannheim GmbH
• sodium acetate trihydrate; NaAc p.a. (M)
• sodium chloride; NaCl p.a.(M)
• Tris ultrapure (M)

Solutions

For the solutions marked with a * a special form (with the protocol on it) is filled out. The other solutions are ready to use or made as described here.

• bloodlysisbuffer:
  • 0.8 x bloodlysisbuffer for 50 ml: poor 40 ml of 1 x bloodlysisbuffer in a 50 ml blue cap tube and fill up to 50 ml with Elga water. Mix by inverting the tube several times
  • 1 x bloodlysisbuffer for 1 litre: poor 50 ml of 20 x bloodlysisbuffer in the marked 1 l bottle, using a 50 ml cylinder. Fill the bottle up to 1 l with Elga water. Mix by inverting the bottle several times.
  • *20 x bloodlysisbuffer for 2 litres, see WFB01: 3.1 M NH₄Cl, 0.2 M KHCO₃, 20 mM EDTA, pH7.4
• EDTA: 0.5 M EDTA (pH8.0), 15575-012, Life technologies
• ethanol: 70% ethanol for 100 ml: add 70 ml ethanol (absolute) to 30 ml of Elga water. Store up to 1 month in a 100 ml bottle
• NaCl:
  • *5 M NaCl for 1 litre, see WFN01
  • *6 M NaCl for 1 litre, see WFN02
• nucleus lysis buffer: *1 x nucleus lysis buffer for 1 l (see WFK01) 10 mM M Tris-HCl, 0.4 M NaCl, 2 mM EDTA, pH8.2
• pronase: *pronase (20 mg/ml) for 50 ml, see WFP01
• SDS: 10 % SDS, 15553-019, Life Technologies
• *TE-4 for 0.5 litre (WFT02): 10 mM M Tris-HCl, 0.1 mM EDTA, pH7.5
• Tris.Cl: 1 M Tris.Cl (pH7.5), 15567-019, Life technologies

Rules for the DNA-isolation shift

General

• write down every abnormal observation on the form (WDNA)
• write down any (accidental) deviation of the protocol (e.g., longer incubation times) on the form (WDNA)
• mark all the tubes during the isolation procedure with the DNA-isolationnr. of the sample; use the printed labels (see LP010).
• you have to schedule the DNA-isolation yourself. There are some rules you have to keep in mind:
  • two tubes of blood from one individual are isolated separately; each tube gets a different DNA-isolationnr: a D1- isolationnr and a D2- isolationnr. The isolation of the D1- and D2-sample of one individual has to be performed in separate rounds of DNA-isolation, e.g. on different days or an isolation round in the morning and one in the afternoon.
  • if just one tube with 8-10 ml of blood from one individual is available, the blood has to be split. 4-5 ml is pipetted into a labelled blue cap tube (D2-nr) and the opened tube (D1-nr) is stored in another labelled blue cap tube.
  • if only one tube with less than 8 ml blood or two tubes both with less than 3 ml blood is available the disease specific technician or a staff member will decide if it is necessary to generate a D2 sample.
• DNA isolation of the D1-sample should be started within 72 hours. If you know the time of blood sampling, check if at least 3 hours have passed, before starting the isolation of DNA (e.g. when the tubes are brought directly from the hospital by a courier)
• do not isolate DNA from more than 48 tubes at once. 48 tubes can be handled in two centrifugation rounds. More tubes will give problems with the incubation times and will give an unwanted time pressure, enhancing mistakes. If you have more tubes plan a morning and afternoon procedure, or perform a procedure the next day.
• prepare the amount of 1 x bloodlysisbuffer that you need yourself. Throw away leftovers from the week before, e.g. 10% SDS and 6M NaCl. Use WLOT ("lotnummerlijst") when you need a new product, e.g. SDS, 99% EtOH, and fill out the forms (WF...) when you make a new solution, e.g. 20 x bloodlysisbuffer, TE-4.
• never pipet directly from a stock-solution, but pour the needed amount in a sterile blue/black cap tube! For every isolation round use a "fresh" tube of 70% ethanol when washing the DNA precipitate.
• for adding nucleus lysisbuffer and ethanol a dispenser is used. Before the first use of the day, discard the first volume from the dispenser. Hold the 50 ml tubes at an angle when you add the solution, so you will not disrupt the pellet (nucleus lysisbuffer) or start the DNA-precipitation too roughly (ethanol).
• if the DNA is not dissolved and more TE-4 is added, write this down on the form WDNA.
• samples that are dissolved on a Friday are stored in the cold room or fridge for the weekend. On Monday the tubes are handed over to the next technician on duty, who puts them overnight on the roller bench. This technician distributes these samples on Tuesday and signs the WDIS (Oracle distribution list) for day 3, after the distribution (see 3.1 below)

Safety measures

• treat all the unlysed blood samples as possible contaminated material. The blood is not checked for the presence of Hepatitis or HIV. Work in the laminar flow and wear protective clothing: gloves and a lab coat.
• however, if it is known that a blood sample is contaminated, the technician who is doing the DNA-isolation has to be informed. He or she has to make a note of this on WDNA. The technician responsible for the diagnostics of this sample has to register this information in the patients file.
• all the tissues, empty blood tubes, gloves etc. that are contaminated with blood are discarded in a yellow biological waste container. The red cell lysate is discarded in the 1 l plastic disposable container.
• you can get a Hepatitis vaccination at the medical service of the AZL.

DNA isolation protocol

1. Day 1: CELL LYSIS
   This part of the protocol is carried out in the laminar flow cabinet in the pre-PCR lab. Wear gloves and a lab-coat.
   NOTE: a blood sample from a baby less than one year old, is lysed with 0.8 x bloodlysisbuffer instead of the regular 1 x bloodlysisbuffer. The relative high amount of fetal haemoglobin in the red cells gives these cells a different osmotic value. Washing of the pellet can be done with the 1 x bloodlysisbuffer.
   NOTE: see LP010 for the procedures concerning sample registration, handling, storage, etc.
   1. print the WDNA list and the labels from ORACLE (see LP010)
   2. check the name and date of birth present on the tube with WDNA, notate any discripancies on WDNA. Label the blood tubes and the empty blue cap tubes with the ‘Oracle-labels’
   3. a second person checks the correct labelling of the blood tubes and signs WDNA.
   4. the volumes in this protocol are based on tubes with 1 to 10 ml of decoagulated blood.
   5. invert the tube several times to get a homogeneous solution. See Troubleshooting if this is not the case.
   6. transfer the blood to a labelled sterile 50 ml blue cap tube and rinse the blood-tube with 1 x bloodlysisbuffer. Add 1 x bloodlysisbuffer to a final volume of 50 ml. Mix gently.
   7. put the tubes on ice for 30 to 60 minutes to lyse the red blood cells.
   8. spin the white cells down for 10 minutes at 1,800 rpm in the centrifuge, use the brake.
   9. Important: check if the lysis was complete before continuing. The pellet should be white and clearly visible, the supernatant should be clear. (see Troubleshooting 2, if this is not the case.)
   10. discard the supernatant in the plastic 1 l container.
   11. rinse the tube with bloodlysisbuffer, until the removal of the red supernatant is complete.
   12. to wash the leucocytes add 15 ml bloodlysisbuffer and put the tube back on ice until centrifugation. Resuspend the pellet just before the centrifugation step. Spin down (see step 1.5) and discard the supernatant. The pellet should be clean, white and without blood-clots. (see Troubleshooting 2)
   13. gently add 3 ml of nucleuslysisbuffer, use the dispenser (see Rules for the DNA-isolation shift, general). Resuspend the pellet with a vortex or tuberack directly before or after the addition of the nucleuslysisbuffer. Do not wait until you have finished a series, because lysis of the leucocytes will start and impair the resuspension of the cells.
   14. add 100 µl of pronase (20 mg/ml) and mix gently. Keep the pronase on ice.
   15. add 300 µl of 10% SDS and mix gently. Check if nothing has been forgotten by looking at the suspension. After addition of pronase and SDS the solution becomes clear and viscous.
   16. if nothing is forgotten and all the samples look well, incubate overnight at 37°C in the incubator or for two days at RT, but not longer than 3 nights. Sign WDNA for completion of day 1
       
2. Day 2: SALT PRECIPITATION
   This part of the protocol has to be carried out in the pre-PCR lab, outside the laminar flow cabinet.
   Check the samples, after the incubation with pronase and SDS, for homogeneity, clarity and viscosity by slowly stirring the tubes "in the light". When a sample is not well lysed, see Troubleshooting 3. If the sample is well lysed, but looks very dirty, you have to inform the technician responsible for the DNA diagnosis of this sample. He or she can than decide to take over the sample at this point and continue with a phenol-chloroform extraction (ALG009)
   1. print the WDIS (Oracle distribution list) list and the labels for the cryotubes from ORACLE (see LP010).
   2. mix the saturated NaCl (6.0 M) stock before use. Add 1 ml of saturated NaCl (6.0 M) to the samples.
   3. shake vigorously for 15 seconds.
   4. centrifuge for 15 minutes at 3.000 rpm in the centrifuge (Heraeus-Christ Varifuge GL), use the brake.
   5. transfer the supernatant to a clean, labelled blue cap tube by "pouring".
   6. repeat step 2.3 and 2.4. If the supernatant isn't clear after 2 centrifugation steps, you should try to remove the debris with a clean pipet, before precipitating the DNA.
   7. precipitate the DNA by gently adding 8 m1 of absolute ethanol (RT), use the dispenser (see par. 9, general). Mix gently and fish out the precipitated DNA with a sealed Pasteur pipet.
   8. wash the DNA in 70% ethanol. Remove traces of ethanol by pressing the DNA against the wall of the tube.
   9. transfer the DNA to a labelled cryotube with 100-350 µl of TE-4 (for 8-10 ml of blood).
      Put a red cap on the cryotubes with a D1-isolationnr. and a yellow cap on the cryotubes with a D2-isolationnr. Write the last three digits of the DNA isolationnr. on the cap.
   10. incubate in the 65 °C incubator for 30 minutes to get rid of any DNase contamination.
   11. put the cryotubes overnight on the roller bench (RT). Sign WDNA for completion of day 2
       
3. Day 3: SAMPLE DISTRIBUTION
   This part of the protocol also has to be carried out in the pre-PCR lab.
   1. put the DNA samples on a bench in the prelab, together with the WDIS. The disease specific technicians collect the tubes there and take over the responsibility for the samples. They sign for receipt of each sample separately, by putting their name, date and initial on the list and store the DNA sample in the fridge. Samples that are not well dissolved are distributed later. The responsibility for the samples ends when they are accepted by the authorized persons. When all samples are distributed, sign WDIS for completion of day 3 and archive WDNA and WDIS in the folder.

References

[1] Miller SA, Dykes DD, Polesky HF (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucl.Acids Res. 16: 1215.

[2] Maniatis, Sambrook and Fritsch: Molecular Cloning. Part 3, 2nd edition: page 9.18.

Troubleshooting

1. if there are coagulations present in the blood or other abnormalities are observed, you should always seek advice from the person in charge (or his/her replecemant). In case of a blood clot it is best to remove it with a Pasteur pipet and isolate DNA from the non coagulated blood that is left over.
2. if a white pellet is not visible, do not discard the supernatant. Always seek advice from the person in charge (or his/her replecemant). There are 2 possible explanations for this situation:
   1. lysis of the red blood cells is incomplete. There is a pellet, but it looks dirty. In this case, resuspend and put the tube back on ice for 15 minutes to complete the lysis
   2. the leucocytes are lysed too. In this case you see a very small pellet. Remove the supernatant carefully with a pipet, and store it until the isolation is completed. Handle the small pellet with care and proceed with the regular DNA isolation protocol.
3. if lysis is incomplete, do not proceed with the protocol. Check if you did not forget to add SDS by shaking the tubes. If no foam formation is seen, add 100 µl of pronase (20 mg/ml) and 300 µl of 10% SDS. If there is foam formation, only add the 100 µl of pronase. Incubate again at 37°C as long as possible. Seek advice.
4. sometimes blood arrives in tubes with glass pearls in it. Get rid of the glass pearls before spinning down the leucocytes. Use a plastic Pasteur pipet to transfer the blood (before or after the lysis) to a clean blue cap tube.

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