SCGI Practicals

• Different types of controls


  • Positive control
  • Negative control
  • No Template control
  • Loading control
• Positive control


  Known DNA sequence containing region of interest
• Negative control


  Nothing should be present on western blot/PCR
• No Template control


  To check for contamination – contains everything except DNA
• Loading control


  Ensures equal amounts of protein have been loaded into wells
• Examples of loading controls


  • B-actin
  • actin
  • a-tubulin
• Common problems with a western blot

  • Unusual/unexpected bands
  • No bands
  • Faint bands/weak signal
  • High background on blot
  • Patchy/uneven spots on block
• Unusual/unexpected bands


  Due to protease degradation
• No bands


  Could be due to many factors including wrong antibody/antigen/buffer used, prolonged washing and low concentration of antibody used
• Faint bands/weak signal


  Low concentration of primary/secondary antibody or too much blocking
• High background on blot

  Too much antibody or old buffers used
• Patchy/uneven spots on block


  Improper transfer e.g. air bubbles trapped between gel and membrane or due to antibodies binding to blocking agents
• In DNA isolation NaCl helps remove proteins bound to DNA
• Bromophenol blue


  Is a dye that helps with gel loading and allows you to track how far gel has ran
• Glycerol


  Makes sample heavier than water so it sinks into the well
• Detergents e.g. triton, tween and NP-40


  Used to isolate proteins
• Coomassie blue

  Produces colour change dependent on protein concentration, reacts with arginine residues and goes from blue to yellow in the presence of proteins
• SDS

  Adds a negative charge to proteins, leading to the unfolding of proteins
• 2B-mercaptoethanol


  Breaks disulphide bridges therefore denaturing the protein
• Heat at 95c


  To break weak hydrophobic bonds
• 3 steps of PCR

  1. Denature DNA (heated to 95c)

  2. Anneal primers (heated to 50c)

  3. Extend primers (heated to 70c)
• Function of blocking

  To reduce non-specific binding of antibody to protein/membrane
• TAE/TBE

  • Establishes pH
  • Provides ions to support conductivity
  • Differ in ionic strength