ANGIOGENESIS – Key to TERM Success All tissues require an adequate blood supply to thrive. TERM researchers continue to explore and manipulate the environment of regenerating tissues, perfecting their ability to predict successful outcomes. Growth factors (VEGF) can be added to enhance angiogenesis, in addition to inherent angiogenesis factor genes. AG genetic polymorphisms vary in potency! 2-fold challenge: 1) Did your company succeed in transforming therapeutic stem cells with a powerful AG gene? 2) Do any of your company researchers possess a powerful AG gene variant? 1

Angiogenesis Gene Search Class Period 1 Student Cell Collection & Lysis Class Period 2 Student DNA Purification Class Period 3 PCR of Student and Forensic Samples Class Period 4 Electrophoresis & Analysis Class Period A PCR of Forensic Samples Class Period B Electrophoresis & Analysis Class Period Electrophoresis of Pre-Made PCR Samples 2

Laboratory ToolsReal-World Applications Learning Objectives Micropipetting Ion Exchange Chromatography PCR: Polymerase Chain Reaction Gel Electrophoresis Human Polymorphic (Hypervariable) Loci DNA Forensics Paternity Testing Medical Analysis Diagnostic Tool 3

Schedule of Class Sessions Session 1: Obtain Cells and Begin Cell Lysis Session 2: Complete Cell Lysis and Column-Purify DNA Session 3: Set Up PCR Reactions on Student and Forensic DNAs Session 4: Run Gel Electrophoresis of PCR Products and Analyze Results 1hr incubation Sessions 2 & 3 can be combined in a double period 1hr 15min PCR reaction 4

Preparing the Lysis Tube Using a marker, label the top of a 1.5ml microtube with the last two numbers of your cell or home phone number. We will call this the “lysis tube”. Add 300μl of First Lysis Solution (ATL) to the tube. Contains detergent to dissolve cell membranes and denature proteins. Lysis Solution can cause burns on skin. Use caution. Report all drips and spills! 5

Option 1: Obtaining a Cheek Cell Sample Carefully rub the flat side of a toothpick along the inside of your cheek for 30 seconds. Swirl the end of the toothpick in your lysis tube until cells are dispersed. Your solution may turn cloudy. Place your used toothpick in the red plastic cup (for biohazardous waste). 6

Lysing Your Cell Sample Close the microtube. Vortex it for 5 seconds. Attach a lid lock to the microtube lid. Place the tube in the 56°C heat block. Incubate for minutes, vortexing briefly every 10 minutes. 7

CLASS PERIOD 2 Finish Purification of Your Own DNA Finish Cell Lysis Ion Exchange Chromatography using Spin-Columns 8

Continuing the Cell Lysis Remove the lid lock. Pulse-centrifuge the lysis tube to remove drops from the lid. Add 300μl of Second Lysis Solution (AL) to the tube. Vortex for 5 seconds. Replace the lid lock and place the tube in the 70°C heat block. Incubate for 5 minutes, vortexing briefly midway through the five minutes. Lysis Solution can cause burns on skin. Use caution. Report all drips and spills! 9

Labeling the Spin-Column and Collection Tubes Open the package containing the spin-column. Label the 2ml round-bottom collection tube below the spin column FT. Label the lid of the spin column with your code number. Label four additional 2ml round-bottom collection tubes in your rack W1, W2, E1, and E2. 10

Preparing the Cell Lysate for Column Binding Remove the lid lock. Pulse-centrifuge the lysis tube. Add 150μl of ethanol to the tube. Vortex for 5 seconds. Pulse-centrifuge the lysis tube. 11

Binding the DNA in the Cell Lysate to the Column Transfer 600μl of the cell lysate from the lysis tube to the center of the paper filter in the spin- column. Don’t touch the pipet tip to the filter. Close the cap and centrifuge at 6000 x g for 1 minute x g = 10,000 rpm or max speed in the Pitt Kit microcentrifuges 12

Washing the Column (First Wash) Transfer the column to the W1 collection tube. Discard the FT collection tube (containing the flow-through) into the red plastic cup. Apply 500μl of First Wash (AW1) Solution to the column without wetting the rim. Close the cap and centrifuge at 6000 x g for 1 minute. 13

Washing the Column (Second Wash) Transfer the column to the W2 collection tube. Discard the W1 collection tube (containing wash #1) into the red plastic cup. Apply 700μl of Second Wash (AW2) Solution to the column without wetting the rim. Close the cap and centrifuge at 6000 x g for 1 minute. 14

Drying the Column (First Ethanol Wash) Transfer the column to the E1 collection tube. Discard the W2 collection tube (containing wash #2) into the red plastic cup. Apply 700μl of ethanol to the column without wetting the rim. Close the cap and centrifuge at 6000 x g for 1 minute. 15

Drying the Column Transfer the column to the E2 collection tube. Discard the E1 collection tube (containing wash #2) into the red plastic cup. Close the cap and centrifuge at 6000 x g for 3 minutes to thoroughly dry the membrane. 16

Preparing to Elute the Column Label the lid of a 1.5ml microtube with your code number and “DNA”. This tube will soon contain your purified DNA. Transfer the column to the labeled microtube. Discard the E2 collection tube into the red plastic cup. Open the lid of the column and incubate at 56°C for 3 min. 17

Eluting (Releasing) the DNA from the Column Apply 100μl of Elution Buffer (ATE) to the column without wetting the rim. Close the cap and incubate at room temperature for 1 minute. Centrifuge at 6000 x g for 2 minutes. Discard the column in the red plastic cup. Store the microtube containing your DNA in a freezer until you perform the PCR. 18

CLASS PERIOD 3 Perform PCR Analysis on Your Own DNA 19

Labeling the PCR Tube With a marker, label the top of a 0.2ml thin-wall PCR tube with your sample’s code number. 20

Preparing the PCR Reaction Reaction ComponentVolume To Add H2OH2O4µl dNTP mix (2mM each)4µl D1S80 Forward Primer (2µM)4µl D1S80 Reverse Primer (2µM)4µl Your DNA Sample4µl 2X Pol + Bfr mix (Taq DNA Polymerase, Reaction Buffer, & MgCl 2 ) 20µl Total Volume40µl 21

Thermal Cycling Place your PCR reaction tube in the thermal cycler. Your instructor will start the thermal cycler running the following thermal profile: Initial DNA Denaturation 95°C for 3 minutes 30 PCR Cycles 95°C for 30 seconds – Denaturation 65°C for 30 seconds – Primer Annealing 72°C for 60 seconds – Strand Elongation Final Elongation 72°C for 5 minutes Cold Storage 4°C overnight (or storage in freezer) 22

CLASS PERIOD 4 View PCR Product DNAs using Gel Electrophoresis 23

Loading PCR Samples onto a Gel The 2X Pol + Bfr in the PCR reaction already has loading dye added. Load 6µl of each PCR sample onto a FlashGel, according to the following chart. Write your sample code in the box corresponding to the lane in which you loaded your sample. Student #1 Student #2 Student #3 Student #4 + AG sample 100bp ladder <- write your sample code here 24

Flash Gel Electrophoresis Conditions 2.2% Flash Gel 200 V 12 minutes 25

ANALYSIS OF D1S80 PCR PRODUCTS 26

Period #_____ Group _____ ldrV CS 1 CS 2 C S1S1 S2S2 2.2% 200V 12min School: Date: 27

Angiogenesis Outcome Interpretation Regarding the DNA Evidence? Regarding each person’s PCR outcome: Certainly Yes? Certainly No? Likely? Unlikely, but Possible? What additional DNA evidence would you need? 100bp ladder Student #1 Student #2 Student #3 Student #4 100bp ladder LeBron Student #1Student #2Student #3Student #4 AG gene 100bp ladder 28

2.2% 200V 12min School: Date: Period _ Group _ V CS 1 CS 2 C S1S1 S2S2 Period _ Group _ V CS 1 CS 2 C S1S1 S2S2 29

C 11 C 10 C 16 C 13 C 15 C 24 C 8.10 C 17 LdrV CS 1 CS 2 C H 11 H 10 H 16 H 13 H 15 H 24 V CS 1 CS 2 CS1S2 Group A Gel 3 - Cheek Group A Gel 4 - Cheek School Date C 11 C 10 C 16 C 13 C 15 C 24 C 8.10 C 17 LdrV CS 1 CS 2 C H 11 H 10 H 16 H 13 H 15 H 24 V CS 1 CS 2 CS1S2 Group A 30