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.

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Presentation transcript:

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).  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.

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

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

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

+ - CBBHBHH

+ - CBBHBHH

+ - CBBHBHH

+ - CBBHBHH

+ - CBBHBHH

+ - CBBHBHH We can convert migration distances into fragment size (kB) by using a standard curve (Size versus migration distance)

+ - CBBHBHH λ 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB 2.0 kB 1.0 kB 0.5 kB Known Measure

+ - CBBHBHH λ 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB 2.0 kB 1.0 kB 0.5 kB Known Standard kbases Distance mm B (mm)BH (mm)H (mm)

Distance Migrated (mm) Size (kBases) 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB 2.0 kB 1.0 kB 0.5 kB Known 4 mm 6 mm 8 mm 9 mm 10 mm 13 mm 19 mm 24 mm Measured Standard Curve Size (Kb)

Distance Migrated (mm) Size (kBases) 6.5 kB 5.0 kB 4.0 kB 3.5 kB 3.0 kB 2.0 kB 1.0 kB 0.5 kB Known 4 mm 6 mm 8 mm 9 mm 10 mm 13 mm 19 mm 24 mm Measured Size (Kb) BBHH mm BBHH mm

H B Plasmid Size = ? kBases 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. Size of this fragment?

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 EtBr incalating)