CSI-TRU Page 1 of 4 Updated: 8/4/2010 DNA Extraction Promega DNA-IQ System Database Protocol Blood card punches Always wear gloves and use barrier (filter) tips when performing this protocol. To prevent confusion and contamination, never process more than 4 blood cards at one time. Preparation: Before beginning, put down a fresh piece of bench paper over the area in which you will be working and then make sure you have all of the following on the work table: Your personal DNA extraction kit Box of gloves (your size) A box of filter tips (small size) Plastic tube rack 1 tube of 1 M DTT from your freezer box (placed in tube rack to thaw) No more than 4 CSI-TRU blood cards (taken from the freezer) Several sterile scalpels Container of sterile toothpicks or pair of tweezers Container of sterile 1.5 mL microcentrifuge tubes DNA-IQ magnetic stand Hole punch 10% bleach solution Biohazard waste container Set of micropipettors Calculator Timer Ultra-fine point sharpie marker Lab notebook Gel pen for writing in lab notebook Also check to make sure that following equipment is available and in working order: Tube heaters (2) with 1.5-mL tube blocks in place Microcentrifuge Vortexer Procedure: 1. Turn on tube heaters: 70 degrees C and 65 degrees C. 2. Calculate the total amount of lysis buffer you will need to prepare by multiplying the number of blood card punches x 250 uL. (E.g. if you are planning to extract the DNA from 4 blood donors today, you will need to prepare 1,000 uL of lysis buffer = 1.0 mL.) 3. Invert the tube of lysis buffer (DNA extraction kit) several times to mix and then aliquot the calculated volume into a sterile 1.5-mL microcentrifuge tube. CSI-TRU Page 2 of 4 Updated: 8/4/2010 4. When the DTT is thawed, mix by vortexing for a few seconds. Then add 1 uL of 1 M DTT for every 100 uL of lysis buffer. When you are done with the DTT, immediately return it to your personal freezer box. 5. Mix the lysis buffer and DTT together by inverting the tube several times. Then aliquot 150 uL of the mixture into each of 4 sterile 1.5-mL microcentrifuge tubes. Be sure to label each of these tubes with the TRU-Sample #. 6. Open the first blood card envelope and use the hole punches to punch out two round blood stained pieces. When you are done, carefully place the 2 blood-stained pieces into the appropriately labeled tube of lysis buffer/DTT. (You may use a sterile toothpick or your tweezers to facilitate this transfer. If you use tweezers, wipe them down with 10% bleach solution between uses.) Then throw away all the packaging and clean the hole punch with 10% bleach solution, making sure that the area that came into contact with the blood card is thoroughly cleaned. Be sure to retain at least half of the blood stain on the card for later use, if needed. The blood card should be stored in its original envelope and placed back in the freezer after use. 7. Repeat step 7 for the remaining blood cards. Be sure to clean the hole punch thoroughly between uses. If you accidentally touch the blood-stained area of a card with a gloved hand, change gloves before processing the next sample. 8. Incubate the tubes in the 70 degree C tube heater for 30 minutes, making sure that the lid of each tube is tightly closed. 9. While you are waiting, prepare your spin basket tubes. Place the required number of 1.5-mL tubes in a rack and seat a DNA-IQ spin basket into each. Make sure that the outside of each 1.5-mL tube is labeled with the TRU-Sample #. 10. At the end of the incubation period, remove the tubes from the heater and transfer the entire contents of each tube (including the blood card punches) into the appropriately- labeled spin basket tube. A sterile toothpick or your tweezers may be used to transfer the blood card punches into the basket. 11. Centrifuge the tubes for 2 minutes at maximum speed (14,000 rpm) in a microcentrifuge. 12. Remove the spin basket from each tube and discard it as biohazardous waste. Then place the tubes in a rack. 13. Vortex your tube of resin (DNA extraction kit) for 10 seconds at high speed or until he resin is thoroughly mixed. Then immediately add 7 uL of the resin to each tube. Do not allow the resin to resettle before adding it to the tubes. 14. Vortex the sample/lysis buffer/resin mixture for 3 seconds at high speed. Then incubate at room temperature for 5 minutes to allow the resin to bind to the DNA. Vortex each tube for 3 seconds every minute during this 5-minute incubation period. CSI-TRU Page 3 of 4 Updated: 8/4/2010 15. At the end of 5 minutes, vortex each tube at high speed for 2 seconds and immediately place it in one of the DNA-IQ magnetic stands. Separation of the beads from the surrounding liquid will occur instantly. If the resin does not form a distinct pellet in the tube, vortex the tube and quickly place it back in the stand. 16. Using a micropipettor with a filter tip, carefully remove and discard all of the solution without disturbing the pellet on the side of the tube. If some of the resin is accidentally drawn up in the tip, expel it and allow re-separation. 17. Add 100 uL prepared lysis buffer to each tube. Remove the tube from the magnetic stand and vortex for 2 seconds at high speed. 18. Return the tubes to the magnetic stand and use a micropipettor to carefully remove all the lysis buffer. 19. Add 100 uL of 1X Wash Buffer (DNA extraction kit) to each tube. Remove tube from the magnetic stand and vortex for 2 seconds at high speed. 20. Return the tubes to the magnetic stand and use a micropipettor to remove all of the Wash Buffer from the tube, being careful not to disturb the pellet. Again, If some of the resin is accidentally drawn up in the tip, expel it and allow re- separation. 21. Repeat steps 19 and 20 two more times for a total of 3 washes. Be sure that all of the solution has been removed from the tubes after each wash. 22. With the tubes in the magnetic stand and the lids open, air-dry the resin for 5 minutes. Do NOT dry the resin for more than 20 minutes, as this may inhibit the removal of the DNA from the beads. 23. Add 100 uL of elution buffer (DNA extraction kit) to each tube. 24. Close the lids and vortex for 2 seconds at high speed. Then incubate the tubes in the 65 degree tube heater for 5 minutes. 25. Remove the tubes from the heater and vortex for 2 seconds at high speed. Then immediately place the tubes in the magnetic stand. The tubes must remain hot as they are placed in the magnetic stand, or yield will decrease. 26. Using a micropipettor, carefully transfer the DNA-containing solution to a sterile 1.5- mL centrifuge tube. Make sure the tubes are labeled with the appropriate TRU- Sample #, followed by the letter "B" (for blood). For example, if you are processing TRU-89, you should label the top and side of the tube "TRU-89-B". 27. Place the tubes (now containing the extracted DNA) in your personal freezer box and place the box back in the freezer. Clean-up: 1. After you are done, be sure to put your DNA extraction kit away. 2. Then discard all debris (used tubes, gloves, used toothpicks, etc.) in the trash. CSI-TRU Page 4 of 4 Updated: 8/4/2010 3. If you used up any items (e.g. beaker of sterile 1.5-mL tubes, box of filter tips), replace them from the supply shelf for the next person. 4. Be sure to turn off the lights and close the door firmly when you leave.