Please enable JavaScript.
Coggle requires JavaScript to display documents.
DNA Extraction & Purification - Coggle Diagram
DNA Extraction & Purification
Extract DNA
Rid of cellular material - proteins, enzymes
Optimal upstream performance - quantification of DNA, PCR
Properties of DNA
Solubility
Density
Electric charge
Base-pairing
Molecular weight
Chelex
Chelating media - binds to cellular components
Removes metal ions from samples
Hb (Haemoglobin)
Samples
Sample Prep
Breaking matrix
Mechanical
Pestle & Mortar
Bead Mill
Vortexing
Heating/Shaking (Thermoshaker)
Enzymatic/Chemical
Proteinase K
Cell lysis & degradation of proteins in forensic DNA extraction
Protease
Cleaves peptide backbone
Dithiothreitol (DTT)
Reduce & disrupt disulfide bonds
, lyses sperm heads :star:
SDS
Separation of proteins based on MW
Lysing cells
Temp
pH
Chemicals/Solvents
Phenol
Chaotropic salts
Detergent (SDS)
Next generation methods - Silica
Silica Adsorption
DNA Extraction Protocol
Bind to silica
in high chaotropic salt conc, low pH
Wash
away contaminants with chaotropic salt
Wash away chaotropic salts with ethanol
Dry
silica membrane/magnetic beads
Elute
in water or low salt buffer, pH 7-9
Magnetic Silica Bead System - BioRobotEZ1
Features "Load & Go" automation of 1-6 samples
DNA in 20 mins
Prefilled reagent strips
Why Automate?
Standardisation
Reliable results
Ease of use
Chargeswitch
Ion-exchange mechanism
Pos surface ligand at low pH & neutral at pH 8.5 & elute DNA
Why not routinely used?
Adv
100% water based
No harsh reagents e.g. chaotropic salts, ethanol
Doesn't compromise efficiency of downstream processes such as PCR
Disadv
Doesn't elute DNA well
Differential extraction
Extract male & female DNA from sample
Sexual assault cases
Sperm cells harder to lyse than epithelial cells
