PRACTICALS

• isolation of genomic DNA:

  • cell lysis produces lysate - cell membrane disrupted to release cell components
  • lysate treated with salt solution to clump together broken components and leaves dna floating
  • centrifugation to separate the pellet from dna
  • dna precipitation - add ice cold alcohol plus salt to produce dna pellet after centrifguation again
• trypsin - pancreatic enzyme that breaks the attachment of the cells with gelatinised plastic
• quantifying DNA and RNA

  • take readings at 260nm and 280nm
  • pure DNA OD260/OD280 = 1.8
  • pure RNA OD260/OD280 = 2.0

  ratio will be less with contamination
• 1 O.D at 260nm for RNA molecules = 40 µg.ml-1 of RNA

  1 O.D at 260nm for single-stranded DNA molecules = 20-33 µg.ml

  1 O.D at 260nm for double-stranded DNA molecules = 50 µg.ml

   
• each complete pcr cycle doubles the amount of dna present
• pcr can be performed on either genomic dna or cdna
• master mix for pcr has all the reagents except for the target dna
• pcr reaction needs taq polymerase, dntps, forward primer and reverse primer and the target dna

  • also add water
• In order to determine whether the PCR has worked and amplified the required amplicon we run the samples on an agarose gel and visualise by staining with Gel red and looking at the gel under a UV light
• Tris-acid solutions are effective buffers for slightly basic conditions. These buffers keep the DNA deprotonated and soluble in water
• EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. But since Mg2+ is also a co-factor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases, its concentration in TBE or TAE buffers is generally kept low (typically at around 1 mM).
• Agarose gels can separate different length pieces of DNA, due to pores formed by agarose. These pores are of the appropriate size to separate DNA of a few hundred to several thousand bases.
• GelRed® is an ultra sensitive, extremely stable and environmentally safe fluorescent nucleic acid dye designed to replace the highly toxic ethidium bromide (EtBr) for staining dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels. The GelRed binds to DNA, when UV light is shown, the intercalated GelRedTM will fluoresce producing a bright orange light.
• tbe contains tris-borate, boric acid and edta
• Western blotting involves the separation of proteins on a polyacrylamide gel, transfer of the proteins to a nitrocellulose membrane, incubation of the membrane with antibodies and finally visualisation of the protein in question
• bradford assay is used to determine the protein concentration within each of the samples
• sds keeps the protein linear
• 2-β Mercapto-ethanol maintains protein activity
• Bromophenol Blue is a pH indicator