PCR using cDNA from undifferentiated ES cells

• Determining whether the PCR has worked and amplified the required amplicon


  1. Run the samples on an agarose gel

  2. Visualise by staining with Gel red

  3. Look at the gel under a UV light
• Agarose gels


  Dissolved in Tris-acid solutions which are effective buffers for slightly basic conditions
• These buffers keep the DNA deprotonated and soluble in water
• EDTA


  • A chelator of divalent cations, particularly of magnesium (Mg2+)
  • Protects the nucleic acids against enzymatic degradation
• Mg2+ is a co-factor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases
• The concentration of Mg2+ in TBE or TAE buffers is generally kept low (typically at around 1 mM)
• What is Gel Red and what is it’s purpose? How does it work? Name two alternatives to using Gel Red

  used to stain dsDNA in agarose gel

  2 alternative = ethidium bromide, methyl blue

  Gel red intercalates with DNA, when a UV light is shone on it the Gel Red dye will fluoresce orange