GEL ELECTROPHORESIS Exercise 5 Announcements Post Lab 5

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 16 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 will take place during your lab period the Week of October 19. There will be 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 March 11.

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 TA teaches Take photos of gel (make 3 copies so you each have one for your notebook assignment) Make sure you understand your photo before leaving

CAUTION UV Light � UV light will damage your eyes

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

- + C B BH H

- + C B BH H

- + C B BH H

- + C B BH H

- + C B BH H

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

Measur e λ C Known 6. 5 k. B 5. 0 k. B 4. 0 k. B 3. 5 k. B 3. 0 k. B 2. 0 k. B 1. 0 k. B 0. 5 k. B + B BH H

- λ C B BH H Known 6. 5 k. B 5. 0 k. B 4. 0 k. B 3. 5 k. B 3. 0 k. B 2. 0 k. B 1. 0 k. B 0. 5 k. B + Standard kbases Distance mm 6. 5 4 5. 0 6 4. 0 8 3. 5 9 3. 0 10 2. 0 13 1. 0 19 0. 5 24 B (mm) BH (mm) 9 8 9 24

Standard Curve Size (k. Bases) Measured Known 10. 0 4 mm 6. 5 k. B 6 mm 5. 0 k. B 8 mm 9 mm 10 mm 4. 0 k. B 3. 5 k. B 5. 0 Size (Kb) 3. 0 2. 0 13 mm 3. 0 k. B 2. 0 k. B 0. 5 0. 3 0. 2 0. 1 4 6 8 10 12 14 16 18 Distance Migrated (mm) 20 22 24 19 mm 1. 0 k. B 24 mm 0. 5 k. B

B BH 9 8 24 mm Size (k. Bases) mm H B mm mm 9 BH mm Measured Known 10. 0 4 mm 6. 5 k. B 6 mm 5. 0 k. B 8 mm 9 mm 10 mm 4. 0 k. B 3. 5 k. B 5. 0 Size (Kb) 3. 0 2. 0 13 mm 3. 0 k. B 2. 0 k. B 0. 5 0. 3 0. 2 0. 1 4 6 8 10 12 14 16 18 Distance Migrated (mm) 20 22 24 19 mm 1. 0 k. B 24 mm 0. 5 k. B

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

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 Et. Br incalating)