Other Gel Systems

• Polyacrilamide System Advantages?

  Useful for proteins

  Synthetic components Higher resolution of smaller fragments
• Polyacrylamide System Disadvantages?

  Prepared like agarose, Neurotoxin (potent)

  Difficult gel preparation (thinner gels)

  There are ready-made polyacrylamide gels
• Advantages of Capillary Gel System?

  Immediate detection

  Increased Sensitivity

  Decreased Labor and Run time
• Disadvantages of Capillary Gel System?

  Very costly

  Molecular biologists are not very well versed
• Carries current and protects samples?

  Running Buffer
• Conjugate base able to take up or release protons to maintain a constant pH (Nucleic acids - pH 8 to 9.5 needs to be maintained).

  Weak acid
• An equation that allows us to predict, to change the pH of the solution with a buffer by 1 unit of pH, you need to dilute used solution to 1/10s of it to the opposite solution.

  Henderson-Hasselbach Equation
• Formula of Henderson-Hasselbach rquation?

  pH = pKa + log [A-]\[HA]
• Increase in buffer concentration = ?

  Increase in heat
• Increase in concentration of the buffer causes increased conductivity and offers greater pH stability.
• Greater conductivity results in greater heat generation at a given voltage. Whatever acid-base added, the pH is steady.
• Too much buffer added?

  Concentration heat is increased
• Buffer conducts electricity - current becomes fast - increased heat.
• Solution of increased buffer = increased heat?

  Higher buffer concentration at a lower voltage.
• Specific Buffers Used

  Tris Acetate EDTA (TAE) buffer

  Tris Borate EDTA (TBE) buffer
• Buffer that is most commonky used for DNA electrophoresis. It migrates faster, and more stable when stored.

  Tris Acetate EDTA buffer
• Buffer that has greater buffering capacity and is prone to precipitation

  Tris Borate EDTA BUFFER
• Buffer Additives?

  Formamide

  Urea
• Buffer additive that lowers denaturation temperature.

  Formamide
• They are commonly used for nucleic acids to break down hydrogen bonds between complementary strands of within the same strand of DNA or RNA. They remain straight.

  Denaturation Agents
• It contains a colored dye and a density agent.

  Loading buffer
• It is used to increase the density of the sample in comparison to the buffer. Very high density chemicals.

  Ex. Ficoll, Sucrose, and Glycerol

  Density Agent
• Monitors the progress of migration.

  Ex. Bromophenol blue

  Tracking dye
• Equipment: Agarose gels ran horizontally in acrylic containers.

  Gel boxes
• Equipment: Electrodes in the gel compartment is connected to a ?

  Power supply
• Equipment:

  Gel is placed in the middle and submerged in the running buffer, filling both compartments.
• Type

  1. Power Supply
  2. Cathode
  3. Anode
  4. Electrophoretic Buffer
  5. Well
  6. Sample
  7. Agarose Gel

  
• Procedure of Gel Loading:

  1. Dilute the samples in loading buffer prior to loading.
  2. Know the capacity of the well (usually 25 uL is a safe volume for a >5 well format -will differ.
  3. Depress the plunger of the micropipette before loading and do not let go until you are done and out of buffer.
  4. Make sure tip is within the well before loading.
  5. When withdrawing the tio, go stright up.
  6. Be cautious

  to not pierce the gel all the way down.
• Detection Systems: After electrophoresis, the bands are visualized using dyes thay specifically associate with nucleic acids (fluorescent dyes and silver stain).
• Fluorescent dyes.

  Silver Stain
• Intercalating agent. Used to be the most widely used dye. Highly carcinogenic. Emits visible loght at 590 nm (orange) when excited at UV light of 300 nm.

  Ethiduim bromide
• Sits in the minor groove of the double helix. Hybridize with double stranded DNA. 25-100 times more sensitive than EtBr. Can be added directly to the gel mix.

  Biggest drawback: (aside from being expensive.) requires special optical filters.


  SyBr Green
• Different types of SyBr Green

  SyBr Green I

  SyBr Green II

  SyBr Gold

  SyBr Safe
• SyBr green that interacts with double stranded DNA (used in RT-PCR)

  SyBr Green I
• SyBr green that interacts with single stranded DNA/RNA staining

  SyBr Green II
• SyBr Green that interacts to both DNA and RNA staining

  SyBr Gold
• SyBr Green type that is a safe variant compared to SyBr green that has been deemed as nonhazardous waste

  SyBr Safe
• It is a fluorescent dye that is intercalating, cannot penetrate cell membrane - only naked DNA/RNA. Has higher sensitivity than EtBr. Can be used for dsDNA, ssDNA, or RNA. Similar absorption and emission spectra as EtBr. Can be used to replace optical imaging system. Can be added directly to gel mix like SyBr green. Cheaper then SyBr green

  Gel Red
• Fluorescent dye that is non-intercalating and nontoxic. Consodered a biohazard. It is more complicated than fluorescent stains. It is increased in sensitivity and it is most useful in protein analysis.

  Silver Stain