DNA Extraction (Omega)

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The protocol for extracting DNA with the Omega Tissue DNA Kit.

Read the entire protocol ahead of time; DNA extraction with this kit takes two consecutive days in the lab but some preparations and arrangements need to be done before day 1 and might take more than a day to be completed, e.g., autoclave materials, order supplies, arrange to use the large centrifuge, etc.

Materials and Equipment

Kits

Omega E.Z.N.A. Tissue DNA Kit - individual spin columns: good for extracting fewer than 8-16 samples or low yield samples. Cheaper per sample (~$1.4-$3.2 per sample).

  • 200 reactions - cat #: D3396-02

  • 50 reactions - cat #: D3396-01

  • 5 reactions - cat #: D3396-00

Omega E.Z. 96 Tissue DNA Kit - 96-well plate columns: good for extracting a large number of samples (saves time by using the multi-channel pipette and in number of centrifugation rounds). More expensive per sample (~$2.1-$2.6 per sample).

  • 4 plates, 384 reactions - cat #: D1196-01

  • 1 plate, 96 reactions - cat #: D1196-00

Reagents and Consumables

Locate/confirm equipment:

  • Gloves - wear gloves to handle all extraction materials.

  • 10% bleach squirt bottle (for sterilizing the bench top)

  • 70-90 % EtOH squirt bottle (for sterilizing the bench top)

  • Razor blades (for cutting tissue)

  • Forceps (for handling the tissue)

  • Lighter (for sterilizing blades and forceps)

  • Weigh paper

  • Kimwipes

  • Calibrated pipettes

  • Autoclaved or sterile/filtered (preferred) pipette tips - sterile/filtered tips are preferred but check with lab manager before you use them.

  • Autoclaved 1.5 eppendorf tubes

  • Tube racks (if using the individual spin columns)

  • Sharps container

  • Trash bin

  • 90% molecular grade EtOH(for sterilizing blades and forceps)

  • 100% molecular grade EtOH in a beaker (for purification if using individual spin columns)

  • Micro-scale (need to be able to measure between 1 and 20 mg)

  • Thermomixer - for the Omega kit, you will use setting #7: “TB Buffer DNA Digest”. It has both single tube and plate attachments, stored in the drawer underneath the counter.

  • Vortexer

  • Microcentrifuge

  • Pipettes (10 uL, 20 uL, 200 uL, 1000uL)

Protocol

Prepping for day 1 of extraction - check list:

  • Calculate how many samples you have in total (including neg and/or pos controls, if any), consider time and cost, and decide whether to use plates or individual tubes.

  • If too many samples to extract in a single round, decide how many samples per extraction batch, remember to leave room for controls if needed.

  • Calculate supplies and volumes needed, and check if you have enough or need to order new.

  • Autoclave materials not provided in the kit (eppendorf tubes and tips (if not using sterile/filtered) and forceps). See autoclave training information if this is your first lab work at the MSC!

  • When using plates, it can be more difficult to keep track of which samples go in which wells, since you can not label wells directly. It is advised to draw the layout of the plate and plan sample allocation on to the wells ahead of time, and keep that layout near by for reference during tissue digestion prep. Also, make sure to keep plates in the same orientation (top left corner A-1) at all times.

Prepping for day 2 of extraction - check list:

  • Reserve plate centrifuge in Hughes lab if using plates.

  • Make sure kit purification buffers are diluted as directed in the kit manual.

Day 1 of extraction - tissue digestion

D1-1. Set thermomixer to desired temperature and rpm (typically 60oC and 450 RPM, but may vary if you are tweaking the protocol).

D1-2. Sterilize surfaces and area around the scale - wipe with with 10 % bleach first, then DI H2O, then 70-90% EtOH.

D1-3. Remove tissue samples from freezer and let thaw (do not let them sit any longer than necessary). Take ~10 samples at the time. Once thawed they can be placed on ice/cold beads.

D1-4. Pre-load lysis plate or sterile eppendorf tubes with 200 uL of TL buffer per well plate or tube using a serile pipette tip.

D1-5. Tare scale with a piece of weighing paper on it.

D1-6. Dip ONLY the cutting edge of the blade into EtOH in beaker. Move the beaker aside. Hold the blade vertically, away from your body and away from the EtOH beaker and flame the blade. When all EtOH has burned off and the flame is gone, set the sterile blade on a clean kimwipe on the bench. Dip the tip of the forceps into EtOH beaker, move the beaker aside. Hold the forceps horizontally, away from your body and from the EtOH beaker and flame the forceps to sterilize.

D1-7. Using the sterile forceps, carefully remove tissue from 2mL tube and set it on the kimwipe. Using the sterile razor blade, cut a piece of tissue 15-20 mg. Pat dry the piece of tissue with a clean kimwipe, so that the tissue appears nearly dry.

D1-8. Place tissue on weigh paper and record the weight in lab notebook. If the tissue weights less than 15mg or more than 20mg, remove or add more tissue as needed to reach this range.

D1-9. Take the weigh paper out of the scale, set it on a clean kimwipe and use the sterile forceps and blade to cut up the tissue into smaller pieces.

D1-10. Transfer the cut up tissue to a well plate or an eppendorf tube, making sure that all pieces of tissue fall into the buffer.

D1-11. Label the tube with sample ID, or keep track of the plate layout on your lab notebook, so you know which samples are in which wells.

D1-12. Discard the used blade into the sharps container. Discard all used kimwipes and wipe the bench again with 70-90% EtOH.

Note: Between samples, a new blade should be used if available and forceps should be sterilized by diping in EtOH and flaming to burn off any residual EtOH and tissue.

D1-13. Repeat steps 5-12 until all samples are in buffer.

D-14. Add 25uL of OB Protease Solution (OB Protease and Proteinase K, provided with the kit, serve the same purpose as digestive buffers) to each sample tube or well plate. You can reuse the tip if you are sure that the tip did not touch the liquid or the sides of the tubes or wells. If the tip does touch any surfaces in the lysis plate or tubes, replace with a sterile tip.

D-15. Close tubes or seal plate with a silicone mat, confirm all tubes or plate wells are labelled correctly and vortex the tubes/plate for 15 seconds.

D-16. Place samples on thermomixer. Before restarting the mixer: Make sure all tube caps are securely fastened/plate is securely sealed. In addition, after 2 minutes on the thermomixer confirm the tube caps/silicone mat have not popped open.

Note: Samples can be left for at least 3 hours or overnight to allow for all tissue to be completely lysed. If possible, pause the mixer, take plate/tubes out and vortex them for 15 sec every 1/2h to 1h to help with lysis.

Day 2 of extraction - DNA purification.

D2-1. Sterilize bench top - wipe with with 10% bleach first, then DI H2O, then 70-90% EtOH.

D2-2. Heat up heating block (or check oven temperature in Hughes lab) to 70oC and place the volume needed of elution buffer (or molecular grade water) in it.

D2-3. Follow the kit manual for plates or individual tubes for DNA purification with the following modifications, if needed:

  • Skip the optional step “add RNAase”

  • Skip the optional step “column equilibration”

  • When centrifuging the lysis plate and the E-Z 96 DNA plate (mini-columns plate), tape them together.

  • Skip the optional step “dry samples at 70oC”

  • We prefer to use molecular grade water as the elution buffer instead of the kit buffer.

  • Elution volume may vary depending on starting material and extraction yield. Minimum volume is 30 uL, to ensure that the entire memembrane is soaked with elution buffer.

  • The second elution can be eluted into a separate tube, if there are concerns about DNA concentration in the first elution.

  • Align the tubes in the centrifuge in this manner:

Tube alignment

D2-4. You will likely need to spot-check the concentration of your extracted DNA, either by Qubit or Picogreen assay. Both are destructive assays, meaning you will need to use some aliquote (typically 1-2 uL) of your eluted DNA to measure concentration. Make note of how much you used from which samples, to keep track of how much is available for downstream library prep and sequencing.

D2-5. Extracted DNA can be place in fridge for a few days, or store at -20oC or -80oC for long term storage. Avoid numerous freeze-thaw cycles.