CHAPTER 1: Some Tools of the Trade Lab 1.2 2014

Become familiar with gel electrophoresis 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

1X SB buffer used in agarose gel solution 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

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

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

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

Magnified agarose matrix

Transfer gels from trays to zip-lock bags 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

Pouring the gels (cont.)

Add buffer to the gel box to just cover the gel–no dimples showing 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

Loading gels Insert pipette tip Under buffer level Above gel well

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

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

Lab 1.2 results A B C

Sample A has blue and purple dye 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 B C

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

Heaviest is purple dye (bromophenol blue) – 670.0 amu 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

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

All DNA molecules have same ratio of charge to mass 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