CSI-TRU Page 1 of 4 Updated: 1/22/2010 DNA Extraction Promega DNA-IQ System Database Protocol Cotton oral swabs Always wear gloves and use barrier (filter) tips when performing this protocol. To prevent confusion and contamination, never process more than 4 swabs 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 reagent freezer box (placed in tube rack to thaw) No more than 4 CSI-TRU buccal swab envelopes (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 Biohazard waste container Set of micropipettors One small beaker for waste 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: Water bath Microcentrifuge Vortexer Procedure: 1. 2. Turn on the water bath to 70 degrees C. Calculate the total amount of lysis buffer you will need to prepare by multiplying the number of swabs x 350 uL. (E.g. if you are planning to extract the DNA from 4 swabs today, you will need to prepare 1,400 uL of lysis buffer = 1.4 mL.) 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. 3. CSI-TRU Page 2 of 4 Updated: 1/22/2010 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 reagent freezer box. Mix the lysis buffer and DTT together by inverting the tube several times. Then aliquot 250 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 #. Open the first buccal swab envelope. If you collected two buccal swabs from your donors, you can use one entire buccal swab in this procedure (and save the other). If you collected only one buccal swab from each donor, you should only take half the swab tip and save the other half in a sterile microcentrifuge tube. Use a sterile scalpel to gently remove the cotton tip. Use the inside of the sterile scalpel envelope as a sterile surface on which to work. When you are done, carefully place the cotton tip in the appropriately labeled tube of lysis buffer/DTT. (You may use the scalpel tip to facilitate this transfer. Then throw away all the packaging and dispose of the scalpel in the sharps container. 5. 6. 7. Repeat step 7 for the remaining swabs. Be sure to use a fresh sterile scalpel for each swab. If you accidentally touch the cotton tip of the swab with your gloved hand, change gloves before processing the next sample. Incubate the tubes in the 70 degree C tube heater for 30 minutes, making sure that the lid of each tube is tightly closed. 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 each 1.5-mL tube is labeled with the TRU-Sample #. Also label an additional 4 microcentrifuge tubes for collection of your final DNA samples in step 26. Make sure the tubes are labeled with the appropriate TRUSample #, followed by the letter "S" (for saliva). For example, if you are processing TRU-89, you should label the top and side of the tube "TRU-89-S". 8. 9. 10. At the end of the incubation period, remove the tubes from the heater and transfer the entire contents of each tube (including the cotton swab) into the appropriatelylabeled spin basket tube. A sterile toothpick may be used to transfer the swab into the basket. At this time, also lower the water bath temperature to 65 degrees C in preparation for the elution step. Centrifuge the tubes for 2 minutes at maximum speed (14,000 rpm) in a microcentrifuge. Remove the spin basket from each tube and discard it as biohazardous waste. Then place the tubes in a rack. 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. 11. 12. 13. CSI-TRU Page 3 of 4 Updated: 1/22/2010 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. 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. Using a micropipettor with a filter tip, carefully remove and discard all of the solution into your waste beaker 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. Add 100 uL prepared lysis buffer to each tube. Remove the tube from the magnetic stand and vortex for 2 seconds at high speed. Return the tubes to the magnetic stand and use a micropipettor to carefully remove all the lysis buffer and discard it into your waste beaker. 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. Return the tubes to the magnetic stand and use a micropipettor to remove all of the Wash Buffer from the tube and discard it into your waste beaker, 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. 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. 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. Add 100 uL of elution buffer (DNA extraction kit) to each tube. Close the lids and vortex for 2 seconds at high speed. Then incubate the tubes in the 65 degree tube heater for 5 minutes. 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. Using a micropipettor, carefully transfer the DNA-containing solutions to the sterile 1.5-mL centrifuge tubes you labeled in step 9. Place the tubes (now containing the extracted DNA) in your personal DNA sample freezer box and place the box in the freezer. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. CSI-TRU Page 4 of 4 Updated: 1/22/2010 Clean-up: 1. 2. 3. 4. After you are done, be sure to put your DNA extraction kit away. Then discard all debris (used tubes, gloves, used toothpicks, etc.) in the trash. 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. Be sure to turn off the water bath and lights and close the door firmly when you leave the lab.