Genomic DNA Extraction
The DNA Sequencing Core offers DNA preparation services for both on- and off-campus clients. DNA prepared by these services is suitable for downstream use with our sequencing and genotyping services as well as other platforms. Genomic DNA extracted by these services has also been validated for high throughput genotyping on the Roche LightCylcer® Platform.
Pricing: price per 100-200 ul sample
|Cancer Center Participating Member||$8.99|
|Cancer Center Associate Members and UCSD Primary Investigators||$11.96|
|Sample Type||Sample Volume||Sample Mass|
|Fresh or Frozen Whole Blood||200 ul||3-12 ug|
|Fresh or Frozen Buffy Coat*||200 ul||25-50 ug|
- We can extract up to 72 samples per day.
- Customer supplies 250-400ul of sample in labeled tube.
- Samples stored over 1 year or at less than -70oC may yield lower quantity and quality of DNA.
- Samples that have undergone freeze-thaw cycles will also affect DNA quantity and quality.
- Please consult with DNA Sequencing Core if samples are not fresh.
- * The buffy-coat fraction from whole blood yields more DNA than an equivalent volume of whole blood.
- To prepare buffy coat:
- centrifuge whole blood at 2500 x g for 10 minutes at room temperature.
- Centrifugation will result in 3 distinguishable
- the upper clear layer is plasma
- the intermediate layer is buffy coat containing concentrated leukocytes
- the bottom layer contains concentrated erythrocytes.
- Carefully collect the intermediate layer and store at -70 oC until ready for DNA extraction.
- Customer will be charged whether or not sample yields DNA. If sample does not yield DNA, we will repeat the assay upon request, but only on the sample that is left in the tube of the original sample. If we are able to extract DNA from the original sample the second time, the customer will only be charged for one extraction. If no DNA yield the second time, the customer will be charged for both extraction assays. Keep this in mind when determining volume of sample to provide.
- To ensure compliance with the National Institutes of Health (NIH) Guidelines, we request that clients provide documentation indicating that the project they wish to submit has been approved by UCSD’s Institutional Biosafety Committee (IBC).
Genomic DNA is extracted from blood lymphocytes using the QiaAmp DNA Blood Mini kit (Qiagen) on the automated QiaCube instrument. The expected yield is 3-12 ug of DNA for extraction from fresh whole blood samples collected with common anticoagulants such as citrate, EDTA, and heparin. A 200 µl sample of whole human blood (approximately 5 x 106 leukocytes/ml) typically yields 6 µg of DNA in 200 µl water (30 ng/µl) with an A260/A280 ratio of 1.7–1.9.
Blood samples are treated with proteinase K and lysed using optimized buffers, and DNA purified using Qiagen Spin column technology. DNA on spin column membrane is washed with Qiagen optimized buffers to remove contaminants. Purified DNA is eluted in buffer (10 mM Tris·Cl; 0.5 mM EDTA; pH 9.0) to prevent degradation.
RNase is not routinely added in the extraction procedure. DNA will be quantified using a NanoDrop.
Approximately 190 ul volume of DNA sample will be returned with two measurements of DNA quantity (in ng/ul) and quality (260/280 ratio to evaluate purity of DNA) performed on a NanoDrop Spectrophotometer. Tubes will be labeled by hand with sample number unless customer provides labels.
Dilution of samples and aliquoting can be conducted at an additional cost.
Samples must be picked up within 2 days of extraction.