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Gel Electrophoresis

Gel Electrophoresis. Exercise 5. Announcements. Post Lab 5 is due before your lab meets next week. Pre Lab 8 will be made available the week of March 17 and due before your Lab 8. LNA DNA/DNA Electrophoresis is assigned today and due next week before Exercise 6 (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 March 17 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 March 17. • There will be a Review Sheet for Exam I posted under Week 7 by March 3. • Exam I covers material from Exercises 1-5. • The last day to drop MCB 151 is March 14.

  3. Week 5 Itinerary • Find your samples from week 4 and store them on ice • Wear gloves (EtBr contamination) • 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 • Dispose of ALL waste properly • Soft EtBr contaminated waste goes in biohazard bags • Gel waste goes in the plastic jug located next to the transilluminator • UV Light • UV light will damage your eyes • Ethidium Bromide (EtBr) • Present in the agarose, gel rigs, benches, etc. • Don’t touch your face or hair while handling. • Take care not to get EtBr on your lab manual or notebook

  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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