chapter 1: some tools of the trade lab 1.2 2014. purpose of lab 1.2 become familiar with gel...
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CHAPTER 1: Some Tools of the Trade
Lab 1.2
2014
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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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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
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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
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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
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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
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Magnified agarose matrix
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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
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Pouring the gels (cont.)
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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
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Loading gels
• Insert pipette tip
• Under buffer level
• Above gel well
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Improper loading technique
• Tip is in the well
• Tip punched through the gel
• Dye spreading under the well
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Proper loading technique
• Tip is above the well and in the buffer
• Sample in well
Courtesy of K. Schramm
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Lab 1.2 results
AA BB CC
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Analysis of sample composition
AA BB CC
• Sample A has blue and purple dye
• Sample B has blue, purple, and yellow dye
• Sample C has blue dye
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Predicted dye molecular weights
AA BB CC
• Heaviest is blue dye (xylene cyanole)
• Middle is purple dye (bromophenol blue)
• Lightest is yellow dye (orange G)
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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
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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
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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