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Gel Electrophoresis

Stay updated for upcoming lab activities, tasks, kits, and exams in DNA gel electrophoresis. Learn about fragment migration, standard curve analysis, and lab safety precautions. Be prepared for Exercise and Exam 1 review.

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Gel Electrophoresis

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  1. Gel Electrophoresis Exercise 5

  2. Announcements • Post Lab 5 is due before your lab meets next week. • Pre Lab 8 will be made available the week of October 20 and due before your Lab 8. • LNA DNA/DNA Electrophoresis is assigned today and due next week before Exercise 6 (Exam 1 Review). • Sign up for Exam I. Exam I will take place during your lab period the Week of October 20. • There is a Review Sheet for Exam I posted under Week 7. • Exam I covers material from Exercises 1-5. • The last day to drop MCB 151 is October 17.

  3. Week 5 Itinerary • Find your samples from week 4 and store them on ice • Wear gloves • Check out kits • Pick up gel • Load and run gel • Take photos of gel (make 3 copies so you each have one for your notebook assignment) • Make sure you understand your photo before leaving

  4. CAUTION • UV Light • UV light will damage your eyes

  5. DNA Gel Electrophoresis • Negatively charged DNA is pulled toward the anode • Large pieces/fragments of DNA do not move as fast as smaller ones

  6. - C B BH H +

  7. - C B BH H +

  8. - C B BH H +

  9. - C B BH H +

  10. - C B BH H +

  11. - C B BH H We can convert migration distances into fragment size (kB) by using a standard curve (Size versus migration distance) +

  12. - λ C B BH H Measure Known 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB 2.0 kB 1.0 kB 0.5 kB +

  13. - λ C B BH H Known 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB 2.0 kB 1.0 kB 0.5 kB +

  14. 4 mm 10.0 6 mm 8 mm 5.0 9 mm 10 mm 3.0 2.0 13 mm 1.0 0.5 19 mm 0.3 0.2 24 mm 0.1 4 6 8 10 12 14 16 18 20 22 24 Standard Curve Size (kBases) Known Measured 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB Size (Kb) 2.0 kB 1.0 kB 0.5 kB Distance Migrated (mm)

  15. 4 mm 10.0 6 mm B B BH BH H H 8 mm 5.0 mm mm mm mm mm mm 9 mm 10 mm 3.0 2.0 13 mm 1.0 0.5 19 mm 0.3 0.2 24 mm 0.1 4 6 8 10 12 14 16 18 20 22 24 9 9 8 24 Size (kBases) Known Measured 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB Size (Kb) 2.0 kB 1.0 kB 0.5 kB Distance Migrated (mm)

  16. Size of this fragment? H You need to make a “map” like this for your plasmid. B • What gel do you expect? (B, H, BH) • Be sure to include plasmid size on your map. Size of this fragment? Plasmid Size = ? kBases

  17. Trouble Shooting (when gel bands are less than ideal) • Consider: • Different amounts of supercoiling • Partial Digests • Small fragments are harder to see (less DNA so less EtBrincalating)

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