Nucleic Acid Isolation Flashcards
What are FIVE types of cellular DNA?
- Genomic (chromosomal)
- Organellar (mtDNA)
- Plasmid (extra-chromosomal)
- Phage / viral (ds or ss)
- cDNA
What are THREE types of cellular RNA?
- mRNA
- rRNA
- tRNA
What are THREE issues with handling DNA and RNA?
- Long and Fragile molecules
- Readily cleaved by nucleases
- Tiny Quantities
Which agent blocks the activity of DNAses?
EDTA
* chelates Mg2+ that DNAses need
Which agent blocks the activity of RNAses?
Diethyl Pyrocarbonate
* do NOT require Mg2+
What are FIVE useful features of DNA molecules?
- Chemically Stable
- Can withstand extreme conditions (pH, salts, solvents etc)
- Folding and Denaturation less important
- Modification less important
- Simple Chemical Composition
What are the THREE goals for NA Isolation?
- Remove proteins and unwanted biomolecules
- Separate DNA and RNA
- Isolation specific DNA or RNA
What are the FOUR main approaches to NA Isolation?
- Differential Solubility
- Adsorption Methods
- Affinity Methods
- Ultra-Centrifugation
What are the typical FOUR steps in the Isolation of Genomic DNA?
- Cell Lysis and Solubilisation
- Phenol-Chloroform Extraction
- Ethanol Precipitation
- Redissolve in TE buffer, add RNAse, then Proteinase K
What chemicals are added during the Cell Lysis and Solubilisation step in the Isolation of Genomic DNA?
Buffer + 0.5% SDS + Proteinase K
How long is incubation and at what temperature is needed at the Cell Lysis and Solubilisation step in the Isolation of Genomic DNA?
1-3 hours at 56°C
What centrifuge speed is required during the Phenol-Chlorofom Extraction step in the Isolation of Genomic DNA?
10,000 G
What does Phenol do during the Phenol-Chlorofom Extraction step in the Isolation of Genomic DNA?
Protein denaturing
What does Chloroform do during the Phenol-Chlorofom Extraction step in the Isolation of Genomic DNA?
Phase Separation
Is High or Low concentrations of salt required at the Ethanol Precipitation step in the Isolation of Genomic DNA?
High
What pH is required at the Ethanol Precipitation step in the Isolation of Genomic DNA?
pH 8
What reagents are used in the Isolation of Plasmid DNA?
- NaOH / SDS
- Lysozyme
What salt is required for the extraction of RNA for RNA Isolation?
Guanidine Isothiocyanate
What pH is required for RNA extraction in RNA Isolation?
pH 5 - 6
What does the Trizol reagent do in RNA Isolation?
Inactivates RNAses
What happens to mRNA during Affinity Isolation with Oligo-dT Media?
Polyadenylated at 3’ end
How many genes does mtDNA have?
37, coding for:
* 2 RNAs
* 22 tRNAs
* 13 polypeptides
Which mtDNA receptor subunit is used in isolating mitochondria?
TOM22
At what wavelength does pure DNA absorb UV light?
260nm
Does dsDNA have a LOW or HIGH extinction coefficient?
Low
Does ssDNA have a LOW or HIGH extinction coefficient?
High
