Flashcards in Lab 6 - Nucleic acids Deck (11):
Nucleic acid groups
-Ribonucleic acids (RNA): contains ribose
-Deribonucleic acids(DNA): contains 2-deoxy ribose
Determination of the conc. of RNA and DNA extracted from the liver
-Separate from free nucleotides and oligonucleotides, by using cold PCA (polynuclotides not soluble). Hydrolyze into bases, pentoses and phosphates in hot PCA. Measue the dissolved pentose.
-Liver from pig, quartz sand, physiological saline, PCA
How to estimate nucleic acid
Pentose after hydrolyzing.
Spectophotometry or UV-photometry.
Abs max(clear RNA and DNA)=260nm
Abs 260/Abs 280: 1.8-2
Determination of RNA by measuring the ribose conc.
-Conc. HCl in orcinol reagent forms furfural from the ribose, with deliberation of 3 H2O.
-Furfural reacts with 2 Orcinol and form a condensation prod.
-Materials:Hot PCA, Orcinol reagent: orcinol+HCl+FeCl3+distilled water, Spectophotometer
Determination of DNA conent by measuring the 2-deoxyribose conc.
-In acidic milieu, 2-deoxyribose is converted into beta-hydroxy-levulinaldehyde by loss of water
Isolation of DNA from pig spleen
-How to separate deoxyribonucleoproteids from other macromolecules?
-How to remove the proteins?
-Must inhibit the DN-ase(deoxyribonuclease) activity, with citratebuffer that binds to Ca2+ and Mg2+(cofactors for DN-ase)
-Deoxyribonucleoproteids are soluble in water and sol. of high salt conc. but insoluble in sol. of low salt conc.
-Removed with chloroform/amylalcohol, and DNA can be ppt. with ethanol.
Cleavage of the A-phage DNA by restriction endonucleases:
-What are restriction endonucleases?
-What are the sequences that restiction endonucleases can regonize?
-How can the DNA fragments be separated?
-What is the restriction endonucleases studied on?
-How can the DNA be seen?
-Enzymes from bacteria that can recognize specific sequences of deoxyribonucleotides with double symmetri-generally 4-8 base pairs, and cut them.
-By gel electrophoresis
-BamHI, HinIII and EcoRI
-UV light, if the agarose contains ethidium bromide - pink.
What is recombination?
A process when DNA fragments are rearanged by the religation, or inserted into the DNA of a foreign organism. Makes it possible to transfer genes from one organism to another.
What are the producing bacteria, recognized sequence and produced sticky end from the restiction endonyckeases?
-BamHI: Bacillus amyloliquefasciens, GGATCC, CTAG
-HindIII: Haemophilus influenzae, AAGCTT, TCGA
-EcoRI: Escherichia coli, GAATTC, TTAA
What are bacteriophages
Viruses that can infect bacteria and replicate in them.