Term
|
Definition
Blood.
Buffy Coat.
Bone Marrow.
Solid tissue.
Lavage Fluids.
Bacteria.
Viruses.
Fungi.
Organelles, mitochondria. |
|
|
Term
| Specimens (Adequate for PCR) |
|
Definition
Dried blood.
Saliva.
Bone.
Teeth.
Amniotic fluid.
Hair follicles.
Buccal cells.
Cerebrospinal fluid.
Fixed tissue.
Feces.
Soil. |
|
|
Term
| DNA from fixed specimens (PCR) - BEST options. |
|
Definition
Acetone.
10% buffered neutral formalin. |
|
|
Term
| DNA from fixed specimens (PCR) - Mediocre options. |
|
Definition
Zambonis.
Clarkes.
Paraformaldehyde.
Formalin-alcohol-acetic acid.
Metharcan. |
|
|
Term
| DNA from fixed specimens (PCR) - Less desirable options. |
|
Definition
Carnoys.
Zenkers.
Bouins.
B-5. |
|
|
Term
| ORGANIC DNA Extraction Methods |
|
Definition
Organic chemicals.
Phenol.
Chloroform. |
|
|
Term
| INORGANIC DNA Extractions Methods |
|
Definition
Inorganic chemicals.
Detergents.
EDTA.
Acetic Acid.
Salt (Salting out, spooling) |
|
|
Term
| Solid Phase DNA Extraction Methods |
|
Definition
| DNA is immobilized on a solid support, beads, or columns. |
|
|
Term
| Speciman Preparation - Bone marrow & Peripheral blood |
|
Definition
Density gradient centrifugation (nucleated WBC)
Differential osmolysis (RBC > WBC)
Streptokinase (clot dissolving) |
|
|
Term
| Specimen Preparation - Tissue |
|
Definition
Freeze/Crush.
Mince.
Enzymatic Digestion. |
|
|
Term
| Specimen Preparation - Plants/Fungi |
|
Definition
Homogenize.
Vortex with glass beads. |
|
|
Term
| Organic DNA Isolation - Steps |
|
Definition
1. Lysis (NaOH, SDS)
2. Acidification (acetic acid, salt)
3. Extraction (phenol, chloroform)
4. DNA Precipitation (ethanol) |
|
|
Term
| Inorganic DNA Isolation - Steps |
|
Definition
1. Lysis (Tris, EDTA, SDS)
2. Protein Precipitation (Sodium acetate)
3. DNA Precipitation (isopropanol) |
|
|
Term
| Solid Phase DNA Isolation - Steps |
|
Definition
1. Lysis (supplied reagents)
2. Acidification (supplied reagents)
3. Adsorption (low pH)
4. Wash (supplied buffer)
5. Elute DNA (low salt) |
|
|
Term
| Solid Phase DNA Isolation |
|
Definition
Solid phase isolation media include silica spin columns and beads.
Nucleic acid binding to silica beads is the basis for many automated extraction systems. |
|
|
Term
| Other DNA Extraction Methods |
|
Definition
Limiting specimens (fixed tissue, dried blood, bone)
Rapid extraction for routine testing. |
|
|
Term
| Rapid DNA Extraction for PCR |
|
Definition
Whole Blood:
1. EDTA or ACD
2. Lysis solution
3. Neutralization
4. PCR
Buccal Cells:
1. Buccal cells
2. Into extraction solution
3. Heat
4. PCR |
|
|
Term
| DNA Purification with Chelex Resin for PCR |
|
Definition
Heat tissue (hair roots, saliva, etc.) in 300 ul of 5-20% Chelex 100 resin.
Chelex removes multivalent cations.
DNA is in supernatent. |
|
|
Term
| DNA Extraction from fixed tissue for PCR |
|
Definition
1. ~20 micron sections
2. Deparaffinize (xylene, ethanol wash)
3. Disgest (proteinase K, Tris buffer) |
|
|
Term
| DNA Extraction/Storage Whatman FTA Paper for PCR |
|
Definition
SPOT: Apply sample and allow to completely dry. Store for future processing or purify as follows.
PUNCH: Remove a disc from the center of the dried specimen.
REAGENT WASHES: Place disc in PCR tube and wash 3 times with FTA Purification Reagent. Discard Reagent after each wash.
TE RINSES: Rinse 2 times with TE-1. Discard TE-1 after each rinse.
DRY: Allow the disc to completely dry.
PCR: Add PCR master mix directly to the disc in tube and PCR. |
|
|
Term
|
Definition
Isolate mitochondria by centrifugation.
Isolate total DNA. |
|
|
Term
|
Definition
Gel Electrophoresis with known standards.
Spectrophotometry:
Concentration: 1 OD260 = 50ug/ml dsDNA
Yield: ug/ml x ml = ug DNA
Purity: OD260/OD280 ~1.6-2.0 |
|
|
Term
Nucleic Acid Extraction
Methods: RNA
Laboratory Preparation:
Bench & Equipment |
|
Definition
| Separate laboratory area designated RNase Free, RNF. |
|
|
Term
Nucleic Acid Extraction
Methods: RNA
Laboratory Preparation:
Disposables |
|
Definition
Certified RNase Free
Rinsed in 0.1% diethyl pyrocarbonate (DEPC) |
|
|
Term
Nucleic Acid Extraction
Methods: RNA
Laboratory Preparation:
Reagents |
|
Definition
Certified RNase free.
Add 0.05-0.1% DEPC (except Tris)
Test with RNase Alert (Ambion) |
|
|
Term
Nucleic Acid Extraction
Methods: RNA
Laboratory Preparation:
Reactions |
|
Definition
|
|
