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CHAPTER 1: Some Tools of the Trade Lab 1.2

CHAPTER 1: Some Tools of the Trade Lab 1.2. 2014. Purpose of Lab 1.2. Become familiar with gel electrophoresis Practice using the micropipette to load wells in practice plates Practice running an electrophoresis gel using three dyes: xylene cyanole , bromophenol blue, and orange G.

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CHAPTER 1: Some Tools of the Trade Lab 1.2

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  1. CHAPTER 1: Some Tools of the Trade Lab 1.2 2014

  2. Purpose of Lab 1.2 • Become familiar with gel electrophoresis • Practice using the micropipette to load wells in practice plates • Practice running an electrophoresis gel using three dyes: xylene cyanole, bromophenol blue, and orange G

  3. SB buffer solution • 1X SB buffer used in agarose gel solution • 1X SB buffer used in electrophoresis box • Materials include 20x SB buffer solution • Dilute 20x SB buffer solution with dH20 to make the 1x SB buffer solution

  4. SB buffer solution procedure • Determine V1, amount of 1x SB buffer needed, by multiplying number of gels by 40 mL • Determine V2, amount of 20x SB buffer used in the dilution, by using the formula (1x)V1 – (20x)V2

  5. Agarose solution • An 0.8% agarose solution is used to make the electrophoresis gel • Materials include agarose • The 0.8% agarose solution must be heated in a double boiler or in a microwave in order for the agarose to dissolve

  6. Agarose solution procedure • Determine amount of agarose solution needed by multiplying number of gels by 30mL • Calculate amount of agarose needed by multiplying number of gels by 0.24g • Carefully heat in double boiler or microwave until all “flecks” are dissolved

  7. Magnified agarose matrix

  8. Pouring the gels • Cool agarose to 60⁰ C and pour into tray until each comb is covered by 2 mm • Once the gels solidify pull each comb straight out of the gel without wiggling • Transfer gels from trays to zip-lock bags • Add small amount 1x SB buffer to gel and store in refrigerator until ready to use

  9. Pouring the gels (cont.)

  10. Setting up the gel box • Play the gel in the gel box so that the wells are at the negative (-) end • Add buffer to the gel box to just cover the gel–no dimples showing • Make sure the gel is in position before loading the wells

  11. Loading gels • Insert pipette tip • Under buffer level • Above gel well

  12. Improper loading technique • Tip is in the well • Tip punched through the gel • Dye spreading under the well

  13. Proper loading technique • Tip is above the well and in the buffer • Sample in well Courtesy of K. Schramm

  14. Lab 1.2 results A C B

  15. Analysis of sample composition • Sample A has blue and purple dye • Sample B has blue, purple, and yellow dye • Sample C has blue dye A C B

  16. Predicted dye molecular weights • Heaviest is blue dye (xylene cyanole) • Middle is purple dye (bromophenol blue) • Lightest is yellow dye (orange G) A C B

  17. Actual dye molecular weights • Heaviest is purple dye (bromophenol blue) – 670.0 amu • Middle is blue dye (xylene cyanole) – 538.6 amu • Lightest is yellow dye (orange G) – 452.4 amu

  18. Dye molecular weight discrepancy • Purple dye (bromophenol blue) has more negative charge per unit of mass than blue dye (xylene cyanole) due to bromine ions • The heavier purple dye molecule travels farther through the gel than the lighter blue dye molecule

  19. DNA molecular weight discrepancy • All DNA molecules have same ratio of charge to mass • The movement of DNA is determined solely by mass and shape • There are 3 plasmid configurations and their shapes affect how far they travel through the gel

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