DNA Extraction
The CGF has established standard operating procedures for DNA extraction to minimize the degradation and contamination of precious research samples.
The CGF offers DNA extraction services for a variety of sample types such as:
- Frozen tissue
- Cells
- Blood/Buffy coat
- Cell free (cfDNA)
- Formalin Fixed Paraffin Embedded Tissue (FFPE) scrolls
- FFPE slides
- FFPE slides with micro-dissection
- For other sample types not listed, please contact the CGF
Sample Requirements
Frozen or FFPE tissue for DNA extraction may be requested through the University of Pittsburgh Biospecimen Core or provided by the individual researcher. Researchers should email the CGF to arrange sample drop-off (insert email address). Samples may only be dropped off after researcher’s have registered though iLab. (Link)
For researchers who are collecting samples on their own, we suggest a few guidelines to limit DNA degradation and ensure sample quality. All samples submitted to the lab for extraction and downstream assays should be handled in one of the following ways at the time of collection:
- Fresh tissue should be flash frozen in 2.0 mL round bottom tubes or Nunc tubes in LN2 or dry-ice and maintained at -80°C until submitted to the CGF. Cells should be pelleted in 1.5-2.0 mL tubes, all supernatant removed, and then flash frozen in LN2 and maintained at -80°C until submitted to the CGF.
- FFPET samples may be submitted in one of two ways:
- Submit two tubes of (5) 10-micron scrolls in each round bottom 2.0 mL microcentrifuge tubes.
*Note: Researchers can request tubes from the CGF if needed prior to collection.
-
- Submit (10) 10-micron blank slides for FFPET extraction. The CGF can perform micro/macro-dissection of the FFPET slides. The researcher must provide an accompanying H&E slide with the area of interest outlined. If this service is requested, we suggest that the researcher talk with a CGF staff member prior to submitting samples to go over the protocol for identifying the area of interest.
Once samples have been extracted, they will be assessed for quality, quantity, and concentration. The CGF will provide a detailed QA/QC report to the researcher.
Fees/Costs
For pricing information, please contact Maureen Lyons.
DNA Services: QA/QC
The CGF offers DNA quality analysis services using the following instruments: NanoDrop ND1000, Qubit Fluorometer, and the Agilent Bioanalyzer 2100.
Researchers can submit the following samples for DNA QA/QC:
- Genomic/mitochondrial/plasmid DNA
- PCR product
- Sequencing libraries
- Custom request
Technology and Equipment
Agilent Bioanalyzer
The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins, and cells on a single platform.
Nanodrop Spectrophotometer
The Nanodrop (Thermo Scientific) is a micro-Volume UV-Vis spectrophotometer for nucleic acid and protein quantitation that allows for sample volumes as small as 0.5 μL. It offers:
Life Technologies Qubit Fluorometer
The Qubit® 3.0 Fluorometer utilizes specifically designed fluorometric technology using Molecular Probes® dyes. These fluorescent dyes emit signals ONLY when bound to specific target molecules, even in the presence of free nucleotides or degraded nucleic acids. Qubit® fluorometric quantitation provides the most specific and sensitive DNA and RNA quantitation available, even at low concentrations.
Other DNA Services
- DNA Library Preparation
- Maureen Lyons.
