Hold micropipet and epitubes at eye level

Micropipet Use 1 Add disposable pipet tip 2 Press plunger to first stop 3 Insert pipet tip into solution to be transferred transferred 4 Slowly release plunger to retrieve liquid 5 Move pipet tip into desired tube 6 Press plunger past first stop to second stop to transfer liquid, keep the plunger down to transfer liquid, keep the plunger down as you remove it from the tube. as you remove it from the tube. 7 Eject tip

Micropipetting technique A

technique B Close the tips!

Micropipet tip should be ABOVE the well NOT IN IT!!!!

Micropipet tip punched right through the gel See dye under the wells

NICE!

When you to into the lab today, you will first practice using the micropipette 15 uL Each person in your group will transfer 15 uL of colored liquid into an empty microtube. Once this is completed, you should have 60 uL of brownish colored liquid in your practice tube.

After practicing how to use the micropipette, a biotechnology student will demonstrate and load the victim’s DNA into the gel. You will then load your DNA into the gels. Each student in the group can then load 1 well of the gel with the suspect’s DNA. There are four suspects total. You may NOT load the gel without the help of a biotechnology student watching you!

To understand electrophoresis, you must understand the structure of DNA

Phosphate groups of nucleotides have a - charge OH PO 4 2- Phosphodiester bond O-P=O - O O 5’ 3’ PO 4 2- OH 3’ 5’ 1 2 3’ 5’

Large groove Small groove 1 Twist = 10.5 bp Charge on a DNA Double Helix 3’ 5’ 3’

Gel Electrophoresis Gel Electrophoresis: technique uses the difference in electrical charge to separate polymers (DNA, RNA, protein) on the basis of size Let’s see a model of how gel electrophoresis works

DNA Electrophoresis analysis after DNA has been cut by enzymes at various points, A and B A B 10 kb 8 kb 2 kb A 7 kb 3 kb B 5 kb 3 kb 2 kb A+BA+B C A B A+B L Restriction enzymes Juang RH (2004) BCbasics

Purpose of Gel Electrophoresis? Get a “DNA Fingerprint” of individuals Trace a sequence of genetic markers in families Diagnose disease Compare genes of different people, different species Find mutations Paternity tests

Restriction Enzymes rDNA (recombinant DNA)—the produced piece of DNA from inserted another piece of DNA recognize specific sites to cut the DNA Blunt ends—straight across Sticky ends—one side of DNA is longer than the other, these overhangs allow for complementary matches between two DNA pieces cut by the same enzyme, the sticky ends match and pasting ma occur to produce an rDNA molecule More than 1200 restriction enzymes discovered & isolated from bacteria Read 5  3 Palindromic (example radar or GAATTC CTTAAG

Reviewing Homework

Based on this DNA Fingerprint created, who should the police arrest as the prime suspect? Suspect 1 or 2? Why?