chapter 1: some tools of the trade lab 1.2 2014. purpose of lab 1.2 become familiar with gel...

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

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

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

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

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.)

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

Improper loading technique

• Tip is in the well

• Tip punched through the gel

• Dye spreading under the well

Proper loading technique

• Tip is above the well and in the buffer

• Sample in well

Courtesy of K. Schramm

Lab 1.2 results

AA BB CC

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

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)

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

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