Biospecimens Accessioning and Processing (BAP) Shared Resource Services

DNA Extraction from Whole Blood

For 10-30 milliliters (mL) of whole blood contained in either anticoagulant citrate dextrose (ACD) or ethylene diamine tetra-acetic acid (EDTA) anticoagulants, services include:

• Logging in and maintenance of sample in database (LIMS)
• Extraction of DNA by Qiagen AutoPure and AutoGen chemistries
• Quantification by UV absorbance and quality control by 260/280 optical density ratio

All DNA samples are quantitated to 250 nanograms/microliter (ng/μL) unless otherwise requested. One 10mL whole blood sample typically yields approximately 350 micrograms (~1.4 mL at 250 ng/μL) of high molecular weight DNA that is 100-200 kilobases (kb) in size.

Long-term storage of DNA samples is available through Mayo Collaborative Services, Inc. (MCSI) in their high capacity ultra cold freezer. DNA samples will be stored by BAP in TE buffer and will be kept as one master tube. The DNA tube is kept at either 4 degrees Celsius or –80 degrees Celsius until pick-up or transfer to long-term storage.

DNA Extraction from Paraffin Embedded Tissue

Certain studies utilize the QIAamp DNA Mini Kit for purification of DNA from most commonly-used human tissue samples, such as muscle, liver, heart, and brain. Paraffin samples are cut into 5 or 10 micron thick sections cut and placed onto slides (not deparaffinized, stained or baked). The TACMA Shared Resource provides paraffin cutting services if needed. Typical DNA yields obtained from various human tissue-sample types using the QIAamp DNA Mini Kit are 15-25ng/μL. Samples can be quantitated and standardized if desired.

DNA Extraction from Frozen Tissue

Frozen tissues are extracted utilizing a phenol/chloroform extraction methodology resulting in highly concentrated, high molecular weight DNA. All DNA samples will be quantitated to 250ng/μL unless otherwise requested.

DNA Extraction from Buccal Cells (Mouthwash Samples)

Buccal cells, collected with a commercially-available mouthwash, are extracted utilizing the QIAmp DNA Mini Kit. Typical yields are 15-25μg/μL. At this time, the Shared Resource does not accept buccal swab brushes for DNA extraction.

Plasma/Serum Separation

Approximately 2-4mL of plasma will be isolated from each 10mL tube of blood contained in either EDTA (for plasma) or no additive (for serum). Plasma isolates are aliquoted in 2mL tubes; however smaller serum/plasma aliquot volumes requiring additional tubes are also available.

Isolation and Cryo-Preservation of White Blood Cells

White blood cells (buffy coat) from whole blood will be isolated, stored in one cryo-vial per 10mL tube. Note: These cells are not being frozen viably. Thus, they cannot be thawed and cultured.

Epstein-Barr Virus Services

Cryo-preservation of viable lymphocytes: white blood cells (buffy coat) from a 10mL tube of heparinized blood are isolated, viably frozen, and stored as cell pellets in three cryo-vials.

Lymphoblast transformation: cell lines are stored in three cryo-vials and frozen in an alarm-monitored liquid nitrogen freezer (unless requested otherwise). In addition, two lymphoblastoid cell pellets are stored for future DNA extraction.

Mycoplasma testing: BAP offers testing of cell cultures and related products for the detection of mycoplasmal contamination.