Systematic Blood Collection for Serum and DNA

Systematic blood collection for serum and DNA

20 ml blood (systematic collection) of blood will be collected from each patient as follows:

Serum collection Material required: 1- BD Vacutainer 10ml SST serum collection Tubes (ref: BD# 367986) 2- BD Vacutainer® Safety-Lok™ Blood Collection Set or other blood collection system. 3- Alcohol swab for cleansing site. 4- Tourniquet. 5- Disposable transfer pipets. 6- Centrifuge capable of generating 1200 RCF(g). 7- Gloves and other personal protective equipment as necessary for protection from exposure to bloodborne pathogens.

Experimental procedure: 1- Draw the blood into two SST serum collection tubes. A trained member of the medical staff should perform this step. 2- For proper additive performance it is important to invert BD SST Tubes 5 times immediately after blood collection. 3- As soon as blood stops flowing in the last tube, remove tube from holder, remove needle from vein, applying pressure to puncture site with dry sterile swab until bleeding stops. 4- Dispose of needle and holder per your facility’s policy and guidelines. 5- Allow blood to clot thoroughly before centrifugation. The recommended minimum clotting time for SST tubes in 30 minutes. 6- Centrifuge the SST tubes for 10 minutes at 1200g. 7- Collect the separated serum into the appropriate collection tubes. Prepare aliquots of 500 ul of serum and store at -80C (or -140C). From two SST tubes you should expect to be able to collect about 12 aliquots (i.e., 6ml of serum).

DNA: Material required: 1- BD Sodium heparin vacutainers collection 10ml Tubes (ref: BD# 367874) 2- BD Vacutainer® Safety-Lok™ Blood Collection Set or other blood collection system. 3- Alcohol swab for cleansing site. 4- Tourniquet. 5- QIAamp DNA Blood Maxi kit (QIAGEN). 6- Centrifuge capable of attaining 4500g, equipped with a swing-out rotor and buckets that can accommodate 50ml centrifuge tubes. 7- Water bath, heated to 70°C. 8- Ethanol (96–100%). 9- Gloves and other personal protective equipment as necessary for protection from exposure to bloodborne pathogens.

1– Draw the blood into one 10ml BD Sodium heparin vacutainers collection tube. 2- Isolation of DNA will be performed using the QIAamp DNA Blood Maxi kit (QIAGEN)

1- Pipet 500 μ l QIAGEN Protease into the bottom of a 50 ml centrifuge tube (not provided). 2- Add 10ml of blood and mix briefly (bring the volume of the sample up to10 ml with PBS, if necessary). 3- Add 12 ml Buffer AL, and mix thoroughly by inverting the tube 15 times, followed by additional vigorous shaking for at least 1 min. 4- Incubate at 70°C for 10 min. 5- Add 10 ml ethanol (96–100%) to the sample, and mix by inverting the tube 10 times, followed by additional vigorous shaking. 6- Carefully transfer one half of the solution from the previous step onto the QIAamp Maxi column placed in a 50 ml centrifuge tube (provided), taking care not to moisten the rim. Close the cap and centrifuge at 1850xg (3000 rpm) for 3min. 7- Remove the QIAamp Maxi column, discard the filtrate, and place the QIAamp Maxi column back into the 50 ml centrifuge tube. Load the remainder of the solution from step 5 onto the QIAamp Maxi column. Close the cap and centrifuge again at 1850 x g (3000 rpm) for 3 min. 8- Remove the QIAamp Maxi column, discard the filtrate, and place the QIAamp Maxi column back into the 50 ml centrifuge tube. 9- Carefully, without moistening the rim, add 5 ml Buffer AW1 to the QIAamp Maxi column. Close the cap and centrifuge at 4500 x g (5000 rpm) for 1 min. 10- Carefully, without moistening the rim, add 5 ml Buffer AW2 to the QIAamp Maxi column. Close the cap and centrifuge at 4500 x g (5000 rpm) for 15 min. 11- Place the QIAamp Maxi column in a clean 50 ml centrifuge tube (provided), and discard the collection tube containing the filtrate. 12- Pipet 1 ml Buffer AE equilibrated to room temperature (15–25°C), directly onto the membrane of the QIAamp Maxi column and close the cap. Incubate at room temperature for 5 min, and centrifuge at 4500 x g (5000 rpm) for 2 min. 3- After purification measure the DNA concentration. You should expect the samples to be at ~ 400ng/ul. 4- Prepare 10 aliquots of 100 ul each and store at -20C (or -80C). Label the tubes with the sample name and the DNA concentration.