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

2. 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

3. 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

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. 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. 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

9. Pouring the gels (cont.)

10. 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

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

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

13. 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

14. Lab 1.2 results A B C

15. 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

16. 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

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