GMO Teaching Supplement for DNA Extraction A photo-guide with useful pointers & tips for a successful DNA extraction Contains photos documenting the key steps in the DNA extraction procedure Photos are numbered according to the BABEC GMO Student & Teacher Guides Can be used for a pre-lab discussion or during lab for troubleshooting & support

5. Internet Exploration: Overview of GMO PCR Lab 1. Collect food or plant product 2. Isolate food/plant DNA 3. PCR of Plant and 35s or Bt genes 4. Gel Electrophoresis GMO- GMO+ 5. Internet Exploration: “All About GMOs” Fall 2011, page 1

Part 2: Removing Impurities Lab Flow, Parts 1 & 2 Part 1: Food Lysis Add plant or food product Add Lysis Buffer Grind Add Lysis Buffer Mix Boil Part 2: Removing Impurities Transfer 400l of liquid Add 40l of NaCl Centrifuge Incubate on ice Centrifuge Fall 2011, page 2

Part 3: Isolating the DNA Lab Flow, Part 3 Part 3: Isolating the DNA Transfer 300l of liquid Add 400l of Isopropanol Mix Centrifuge Remove liquid Resuspend DNA in TE/RNase Dry DNA pellet Store on ice Fall 2011, page 3

Use a Small Amount of Food Material The portion of food/plant material should sit about halfway to the 0.1ml mark (step 2) 0.1ml mark The correct amount of corn meal Fall 2011, page 4

Food Maceration Add 200l Lysis Buffer to food material (step 3) Crush with micropestle (step 4) Twist and grind with an up & down motion Keep food debris between pestle and tube wall If necessary, remove stuck portion from bottom of tube with pipette tip Add 800l Lysis Buffer to crushed material (step 5) Solid material may still remain, but liquid should become cloudy Fall 2011, page 5

Ideal Centrifuging Method Orient the hinge of the tube to point outward and away from the middle of the centrifuge. This allows for solid material to settle to the bottom of the tube directly below the hinge. Hinge of tube points out All spins should be performed at 10,000 x g, which is about 10,000 rpm depending on the centrifuge If you have a less powerful centrifuge, spin longer than 5 minutes below hinge side view After centrifuging, look below the hinge for the solid material (pellet) Fall 2011, page 6

Removal of Food Lysate Lysate is the solution produced when cells are destroyed. You want to take the clear liquid, which contains the DNA, not the insect debris. After centrifuging, there may be food debris on bottom of the tube, and possibly on the top Place your pipette tip between the two layers and take 400l of the clear liquid (step 9) Lipids float on top Take liquid from here Proteins settle to bottom Fall 2011, page 7

Incubation on Ice The NaCl precipitates the SDS in the lysis buffer. SDS can degrade DNA so you want to remove it. Food lysate (400l) with NaCl added (40l) (step 10) After sitting on ice, the NaCl solution may become cloudy because a precipitate has formed ice Fall 2011, page 8

Isolating the DNA After the SDS precipitates out, the DNA is in the supernatant, the liquid portion. After you centrifuge, the solid material settles to the bottom. Transfer 300l of the liquid into a new tube and add 400l isopropanol (step 13 & 14) DNA is in the liquid SDS; discard Fall 2011, page 9

Drying the DNA Pellet After the final centrifuge spin, the DNA is now in the bottom of the tube because it precipitates with isopropanol Pour off the liquid & dry the pellet (steps 16 & 17) You may or may not see a pellet. DNA is still there even if you can’t see anything. Here are some examples of what you may see: No visible DNA pellet Small DNA pellet Large DNA pellet Your DNA is here, directly under the hinge Fall 2011, page 10

Resuspending the DNA Depending on how large your pellet was, your DNA may be clear or cloudy. DNA is resuspended in 200l TE/RNase (step 20) If DNA is cloudy or if there is visible debris, add another 200l (step 21) Cloudy DNA DNA with debris Clear DNA Excessive food debris can inhibit the PCR and it helps to dilute it out. You should still have plenty of DNA. Fall 2011, page 11