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The Suspects 1.Ms. Hall 2.Clarence 3.Eduardo (Gertrude’s Boyfriend) 4.Britney (Gertrude’s best friend) 5.The Janitor

But how do we solve this murder???????

1.Collect samples from the crime scene and the suspects 2.Isolate DNA from the samples 3.Use PCR to make lots of copies of section of the DNA 4.Cut up the DNA fragments using Restriction Enzymes 5.Run on a gel electrophoresis and compare banding patterns

Our task We need to determine if the DNA from one of our suspects matches the DNA found at the scene of the crime Which one could have killed Gertrude….

Day One

SAFETY PROCEDURES 1.WEAR YOUR GOGGLES AT ALL TIMES! 2. DO NOT EAT FOOD DURING THE LAB 3. DO NOT CONSUME ANY LAB MATERIALS AND EQUIPMENTS

Lab Procedures 1. WEAR YOUR GOGGLES AT ALL TIMES!!!!! 2. Label EVERYTHING!!! (lab name, date, lab period) 3. Add 10μL of the Restriction Enzyme (ECORI and PStI) in each DNA sample 4. Mix the DNA and ENZ 5. Incubate at 37°C for 45 minutes (we will do this together so STOP before you get to #5) 6. After incubation, keep your DNA samples in the refrigerator

JOBS ❖ Material Manager 1. Get the materials before the Lab 2. Make sure materials are correctly placed at the end of the Lab ❖ Safety Inspector 1. Makes sure that every scientists are wearing their goggles 2. Make sure the lab area is cleaned up after the Lab ❖ Recorder 1. Label EVERYTHING! Including waste beakers, DNA samples 2. Responsible for all the data collecting ❖ Questioner 1. Responsible for asking questions to the teacher (only one allowed to ask questions about directions, procedures, etc)

Micropipette rad.com/webroot/web/html/lse/support/tutori al_micropipet_wndw.html

Things to AVOID !! 278 Do NOT use the pipette in a vertical orientation.

Things to AVOID !! 278 Never use a pipette without a tip. NEVER EVER REUSE A PIPETTE TIP FOR A NEW SAMPLE

Restriction Endonucleases aka restriction enzymes

What did you do today? What is the purpose of adding the restriction enzymes? What were the TWO restriction enzymes we used today? Where did the the DNA samples of the suspects and the victim come from? what other sources could we have used? Think About It !

What are DNA restriction enzymes? Bacterias Immune System! Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands Recognize very specific sequences Over 3,000 known enzymes

Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4-6 base pairs, palindromic sequences (eg GAATTC) Palindrome Restriction site Fragment 1 Fragment 2

Sticky ends vs. blunt cut Sticky Ends Blunt ends -Sticky ends: when a restriction enzyme cuts near the beginning or end of the restriction sequence, leaving unpaired nucleotides -Blunt cut: when the restriction enzyme cuts directly in the middle of the restriction sequence

Sticky Ends Enzyme cuts -They’re called “sticky” because they pair with other complementary sticky ends Good: These sticky ends can be useful when inserting DNA into a plasmid (bacterial genome). Bad: In PCR, these sticky ends will pair with other sticky ends, binding your DNA fragments together.

Blunt cuts Blunt cuts don’t reanneal (stick back together) as easily as sticky ends. This is very good for PCR, because your DNA fragments will stay separated.

Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

Let’s Practice !!! Restriction Enzyme Activity!

Exit Ticket Questions 1.What is a restriction enzyme (what does it do)? 1.What would happen if somebody forgot to change the pipette tips for each one?