Molecular Hybridization Flashcards
Hybridization
when 2 complementary strands of DNA anneal to form Double stranded DNA molecule. It is not renaturation because it is not one double stranded molecule being separated then brought back together
Tm is dependant on?
ionic force, base composition, length, Ph >12, presence of helix-destabilizing agents, DNA mismatch
what effect do urea and formamide have on DNA
they both form H bonds with the DNA bases, saturating the H bond sited and preventing the formation of inter-base bonds
high stringency conditions lead to?
extremely perfect base pairing . it is when formamide is at 42’
low stringency conditions lead to?
imperfect base pairing of a few strands, but still stable. is at formamide at 35’
electrophoresis
allows the rapid and precise separation of molecules in a wide range of dimensions
electrophoresis on agarose gel
is a polysaccharide that forms pores of various size, between 100-300 nm diameter. it acts like a sieve. Speed depends of dimension
electrophoresis on polyacrylamide gel
is composed of monomeric chains of acrylamide cross-linked with units of NN’- methylene bisacrilamide .The pore size depends on concentration of acrylamide and on ratio of acrylamide and bisacrilamide
DNA fragments migrate with mobility inversely proportional to
log10 of their molecular weight
a calibration curve is used to
determine the length of a fragment on the basis of its relative mobility in gel electrophoresis
what is southern blotting
Southern blotting: is used to transfer DNA from the gel onto a filter which is a nylon or cellulose membrane.
what is northern blotting
Northern blotting: is used for RNA transfer
what is western blotting
Western blotting: is used for protein transfer. It uses an antibody to recognise proteins
what is far western blotting
Far western blotting: uses glycogen protein to recognise proteins
what is eastern blotting
Eastern blotting: uses proteins to detect post-translational modifications on proteins
