Gel electro

• Polymerase chain reaction (PCR)


  Technique to amplify specific DNA fragments without the need for bacterial cells
• Taq polymerase


  • Heat-stable DNA polymerase originally isolated from Thermus aquaticus, a bacterium that lives in hot springs at temperatures above 90°C
• PCR process


  1. DNA sample mixed with Taq polymerase, deoxyribonucleotides, and primers

  2. Heated to 95°C to separate DNA strands

  3. Cooled to 60°C to allow primers to hybridize to target DNA

  4. Temperature raised to 72°C for polymerase to add nucleotides

  5. Temperature raised again to separate new and original DNA strands

  6. Cooled to allow primers to bind again to target DNA, now doubled in amount

  7. Cycle repeated to exponentially amplify target DNA
• Gel electrophoresis


  Technique to separate nucleic acids of different molecular mass (i.e. nucleotide length)
• Polyacrylamide gel electrophoresis


  • Used to separate small RNA or DNA molecules of a few hundred nucleotides or less
• Ethidium bromide


  Stain that intercalates into the DNA double helix and causes DNA bands to fluoresce under UV light
• PCR reagents


  • Distilled or deionized H2O (sterile)
  • Primer A
  • 10X PCR buffer
  • Primer B
  • dNTPs (2 mM)
  • DNA template
  • Taq polymerase
  • MgCl2 (if not in buffer)
• Gel electrophoresis materials


  • Agarose gel
  • Paraffin film
  • Loading Dye
  • Gel electrophoresis system
  • Running buffer
  • 1X TBE (890 mM Tris-borate, 890 mM boric acid, 20mM EDTA)
  • Grl green
• PCR procedure


  1. Mix reagents in PCR tube

  2. Run in thermal cycler with specified conditions
• Gel preparation

  1. Weigh 1 g agarose and add to 100 ml 1X TBE

  2. Boil until dissolved

  3. Add 3 µl Gel Green

  4. Pour into gel receptacle and place comb
• Gel electrophoresis


  1. Mix PCR amplicons with loading dye

  2. Place gel in electrophoresis chamber with buffer

  3. Load samples and DNA ladder

  4. Run at 135V for 18 min or 100V for 30 min

  5. Visualize gel under UV light
• DNA (negatively charged) migrates towards the positive electrode (anode) through the gel matrix, with smaller fragments moving faster due to less resistance in the pores
• Gel can be stored at 4°C in 1X TBE buffer wrapped in Saran wrap to prevent drying
• DNA amplification is important to generate several copies of a DNA sequence for further analysis
• DNA ladder


  Reference for size estimation of DNA fragments in gel electrophoresis
• If PCR samples do not separate on the agarose gel, it could be due to issues with the gel preparation or running conditions
• Polymerase chain reaction (PCR)


  Method to amplify DNA fragments without bacterial cells, using Taq polymerase.
• Taq polymerase


  Heat-stable DNA polymerase from Thermus aquaticus, used in PCR.
• Oligonucleotides


  Short synthetic DNA fragments serving as primers in PCR.
• Thermal cycler


  Instrument automating temperature changes for PCR cycles.
• Agarose gel electrophoresis


  Technique to separate nucleic acids by size using a gel matrix.
• Ethidium bromide


  Stain intercalating DNA, causing fluorescent bands under UV light.
• DNA ladder


  Reference standard of DNA fragment sizes in gel electrophoresis.
• Micropipettors


  Instruments for precise liquid handling in small volumes.
• Polyacrylamide gel electrophoresis


  Method to separate small RNA or DNA molecules by size.
• Gel Green


  Dye added to agarose gel for visualization during gel electrophoresis.
• 1X TBE buffer


  Buffer solution for gel electrophoresis containing Tris-borate and EDTA.
• DNA amplicons


  PCR products generated by amplifying specific DNA regions.
• Electrophoresis chamber


  Container for running gel electrophoresis experiments.
• Saran wrap


  Plastic film used to cover and prevent drying of stored gels.
• PCR cocktail


  Mixture of reagents used in polymerase chain reaction.
• Running buffer


  Buffer solution in the electrophoresis chamber for DNA movement.
• Gel electrophoresis system

  Equipment setup for separating DNA fragments in a gel.
• Microwave


  Appliance used to dissolve agarose when preparing a gel.
• Gel comb


  Tool to create wells in agarose gel for loading samples.
• DNA template

  Original DNA sample or bacterial colony used in PCR.
• Magnesium chloride (MgCl2)


  Additive sometimes used in PCR buffer for optimal enzyme activity.
• Grl green


  Typo likely referring to Gel Green, a dye for visualizing DNA in gels.
• PCR machine


  Instrument for running polymerase chain reaction cycles.
• Loading dye


  Dye mixed with DNA samples for loading into gel wells.