Professional Documents
Culture Documents
Nucleic-Acid Isolation Methods: Michael T Madziva, PHD
Nucleic-Acid Isolation Methods: Michael T Madziva, PHD
Nucleic Acids
DNA RNA Genomic DNA Plasmid DNA
Animals
Our method (Blin & Stafford, 1976) should yield DNA of 100-150 Kb; adequate for Southern analysis and construction of genomic libraries
Formamide & dialysis (Kupeic et al. 1987) gives higher MW DNA but final concentration very low. Have quicker methods for applications that dont require high MW DNA eg Bowtell 1987 guanidine to disrupt cells, 80 kb product
Add to aqueous phase 1 vol of chlorofom, mix and spin (to remove phenol traces) EtOH precipitate DNA to concentrate and remove traces of phenol/chlorofom
Precipitation of DNA
Salt and EtOH (ion pairs ppt, EtOH reinforces) Add 1/10 vol. (relative to aqueous) of 3M Na acetate pH 5.2 Add 2-2.5 vol EtOH or 1 vol isopropanol Spin Wash with 70% EtOH Alternatively: Add 2 vol. 7.5M ammonium acetate (NH4+ - free nucleotides in soln) 2 vol. EtOH or 1 vol. isopropanol Spin Wash with 70% EtOH
Critical Parameters
Need to minimize activity of endogenous nucleases
Freeze tissue EDTA in solubilization buffer Minimize shearing of DNA Gentle (but thorough) mixing Avoid EtOH pptn and instead remove organic solvents and salt from DNA by dialysis
Purity : index A260/A280 high quality DNA ratio larger than 1.8 Ratio of 1.5 indicates soln of 50% DNA 50% protein
Summary (cont)
In general 1g tissue or 109 cells can yield 2mg DNA
DNA should be at least 100kb long Very viscous solution
DNA can be stored indefinitely in the presence of EtOH at 4OC or in TE buffer at -20OC TE buffer is a pH buffer & base hydrolysis catalysed by divalent ions
For MIDI or MAXI preps, can ppt DNA with polyethylene glycol
Isolation of mRNA
Required for gene cloning and expression analysis Major difficulty in RNA isolation is that most ribonucleases (RNases) are very stable and active enzymes that require no cofactors to function Therefore, first step in RNA isolation is to lyse cells in the presence of chemicals that will denature ribonucleases Crucial that denaturant in contact with cellular contents at moment of disruption as RNA unstable as harvest begins
Isolation of mRNA
All solutions DEPC (diethylpyrocarbonate) treated Lyse in the presence of RNase inhibitors eg placental ribonuclease inhibitor (RNAsin) Lyse cells and release cytosol, pellet nuclei/membranes, then phenol extract and EtOH pptn
If have contaminating DNA can remove with RNase-free DNase I RNA suspended & stored in safe RNase-free soln
References
Blin, N. & Stafford, D.W. 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303. Bowtell, D.D.L. 1987. Rapid isolation of eukaryotic
DNA. Anal. Biochem. 164, 53. Kupeic et al. 1987. Isolation of high molecular weight DNA from eukaryotic cells by formamide treatment and dialysis. Anal. Biochem. 164, 53.
mmadziva@curie.uct.ac.za