Tools and techniques Flashcards
Basic requirements of nucleic acids isolation
Disruption, separation/isolation/deproteinisation, purification
Detergent for plant cells
CTAB
Detergent for animal, yeast, bacteria and fungi cells
SDS
Reactants used for deproteinisation of plan cells
Chloroform/isoamyl alcohol
Reactants used for deproteinisation of animal and bacterial cells
Phenol/chloroform
Once deproteinisation occurs, where’s located nucleic acids?
In the supernant, acquous part.
If you want to get only mRNA what should you add?
oligo dT-cellulose
When precipitating genetic material, what do you add to the material?
Cold isopropanol (or ethanol), with high concentration of salt, for protecting DNA. Then washed with 70% Ethanol and washed with TE (Tris EDTA) to takes out the metal of the salt. And fially to get rid of RNA, and RNase.
What’s a chelant molecule, example and how it helps you when precipitating nucleic acids?
This molecule takes metals, example TE, and helps you to take out metals from salts previously added.
Method used for mRNA purification
Affinity chromatography
Method used for plasmid DNA purification
Gradient centifugation
Chemical used for pDNA purification
CsCl
For long storage of DNA, what buffer is recommended?
TE (Tris-EDTA)
Absorbance of of dsDNA at 260nm
50microg/mL
Absorbance of of ssDNA/RNA at 260nm
40microg/mL
Ratio of wavelength for determining purity
260/280 nm between 1.8-2
Isotopes used for dNTPs at radiolabelling
3H, 32P, 35S, 14C
Differece between probing and tracking
Specificity
Radiolabelling that uses Polynucleotide kinase
End labelling
Purines
A y G
Pirimidines
T y C
What does PNK does?
In end labelling, puts a radioactive P onto the 5’ hydroxi termini
What enzyme is used in nick translation
DNA Pol. I
How do you create a nick in the DNA
Low concentration DNase
