DNA Isolation Methods.

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Presentation transcript:

DNA Isolation Methods

DNA Isolation Methods Easiest: direct use without isolation Boiling method Phenol/chloroform extraction Alcali-lysis method Commercial kits according to instructions

Which DNA method? How to decide? Total time and labor for the job Reliability of isolation method regarding diagnosis Contamination risks Isolation of sufficient amount and pure DNA for amplification As the number of processes increases DNA losses also increses

Commercial DNA Isolation Kits Genomic DNA isolation kit (Fermentas) DNA isolation kit for blood/bone marrow/tissue (Roche) High Pure PCR Template Preparation Kit (Roche) DNeasy Tissue Kit / QIAamp DNA Mini Kit / QIAquick PCR Purification Kit (Qiagen) MasterPure™ Complete DNA and RNA Purification Kit (Epicentre) 200-400 € prices

Storage of Isolated DNA In DNA TE (Tris-EDTA, pH 7.4-8.3) buffer, DEPC (diethylpyrocarbonate) treated water or just in steril water. Although DNA can be stored in room temperature For daily storage at 4ºC For long-term storage at -20ºC For longer storage (years) at -70ºC. Do not forget that DNA has a fragile structure. Be careful not to freeze-thaw DNA a lot since it can cause mechanical destruction of DNA

Problems faced with DNA isolation

These are; Cross-contaminations External (environmental) contaminations Loss of DNA due to wrong manupilations Insufficient DNA isolation