Gel Extraction (QIAquick Extraction Kit protocol here updated with extra tips!)

Preparing the Gel

  1. Dissolve agarose to a final concentration of 1%(by mass) in TAE buffer in a glass bottle.
  2. Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously. ~1 minute/200ml solution
  3. Place the solution in a warm water bath for 5 mins.
  4. Add 10 µl SYBRSafe (1:10000) per 100 ml of the solution and mix well.
  5. Pour 50ml of solution per small gel tray. (the gel trays and combs should be pre-cleaned with water and wiped dry).
    • Note for combs: 15-well combs hold about 6 ul liquid, 12-well combs hold about 15 ul, 8-well combs hold about 20 ul
      • Taping two 8-well comb wells together results in a well that holds up to 100 ul
      • Taping three 8-well comb wells together result in a well that holds up to 200 ul
  6. Use 120ml per large gel tray. [need to update amounts]
    1. For the small set: small trays hold 20ml, large trays hold 50ml
  7. Wait for the gels to solidify. ~15 mins
  8. Label and store at 4C.

Running the Gel

When doing gel extraction, it is important to run both an analytical gel (to view under UV) and an extraction gel (from which bands are excised). UV damages DNA, and so we dont want to expose our extracted DNA.

Analytical Gel:

The analytical gel should have between 20 and 100 ng of DNA in each well. It should be an exact copy of the extraction gel with respect to position, voltage, and run time.

Extraction Gel:

This should be the rest of the digestion(s).

The analytical and extraction gels can technically be part of the same physical gel. Make sure to separate with a razor blade before imaging.

Refer to Gel Prep protocol above to determine the amounts of liquid to load for the specific well.

Appropriate Hyperladder to be used for PCR product which is linear. Usually Hyperladder I will be used.

  1. While casting gel, add two sets of lanes; use one set to load an analytical gel. 
  2. Add 2ul gel loading buffer (Orange G 6X; it helps DNA sink into the bottom of the well) to DNA.
  3. Make sure there is enough 1xTAE in the plate holder.
  4. Load 5.0ul of appropriate hyperladder to one of the lanes.
  5. Load appropriate amount of DNA - As much as possible! Usually 15-18ul - (mixed with the buffer) in each well.
  6. Set the timer and voltage to 100V and 25 min.

Analytical Gel Annotation

The following things need to be added to the analytical gel image BEFORE it is posted to the wiki:

  • Label each lane with part number and amount of DNA loaded
  • Label each band with length and proposed identification
  • Include wt% agarose, run time, and voltage

Gel Extraction Protocol using Zymo kit (preferred if available)

  1. Place the extraction gel on the blue light table.
  2. Cut out the appropriate bands. Place into 2mL microtube(s). Try to cut out as small a piece as possible while still getting all the DNA.
  3. Weigh gel slice (tare with empty microtube). Add 3 volumes of ADB buffer per mg of gel (so a 100mg gel gets 300 uL of ADB buffer).
  4. Incubate at 55C for 10 minutes. Make sure that the gel is completely dissolved.
  5. Add dissolved gel solution to Zymo column in collection tube. Max volume is 800 uL at a time.
  6. Spin 14000 rpm for 30 sec.
  7. Discard liquid in collection tube.
  8. Repeat step 5-7 if had more than 800 uL dissolve gel.
  9. Add 200 uL DNA wash buffer.
  10. Spin 14000 rpm 30 seconds.
  11. Discard liquid in collection tube.
  12. Add 200 uL DNA wash buffer
  13. Spin 14000 rpm 1 min.
  14. Discard liquid in collection tube.
  15. Spin 14000 rpm 1 min one more time (dry spin).
  16. Discard collection tube (but not the column).
  17. (Optional: 2nd dry spin into clean collection tube.)
  18. Place column in a clean labeled microtube.
  19. Add 10 uL (min 6 uL for higher DNA concentration) of sterile DDH2O to top of column. Water should be pipetted directly onto center of filter.
  20. Incubate at RT 1 min (or longer).
  21. Spin 1 min at 14000 rpm. Discard the column.
  22. Measure the concentration on the nanodrop. (You may recover the 1uL from the nanodrop if needed.)

Gel Extraction Protocol using QIAquick Gel Extraction Kit:

  1. Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator. 
  2. Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV. 
  3. Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue. 
  4. Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
  5. Add 3 volumes (6 volumes if you are afraid of getting a low yield) of Buffer QG to 1 volume of gel (100mg ~ 100ul)
  6. Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
  7. Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
  8. Add 1 gel volume of isopropanol
  9. Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT)
  10. Run flow-through over column one more time.
  11. After the second time, discard flow-through and place column back in tube.
  12. If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
  13. Add 500uL of Buffer QG to column and centrifuge for 1 min (wash).
  14. Wash: add 0.75ml Buffer PE (make sure that the buffer has ethanol added to it) to column.  Let stand for 2-5 minutes and then centrifuge for 1 min
  15. Discard flow-through & centrifuge for 1 min
  16. Place column into clean Eppendorf tube
  17. Add 50ul Buffer EB or water to center of membrane.  Make sure to use warm EB (50C).  (Use 30uL if worried about low concentration.)
  18. Let stand at RT for 4 min
  19. Centrifuge for 1 min
  20. Measure the concentration using the UV spectrophotometer.
Pro Tips
  1. You don't need 2 lanes if you aren't putting your gel under UV light (the blue light and SYBR safe is fine)
  2. You can up the IPA to 1/4 of the total volume
  3. Warm EB (50 mL conical filled w/ water, plop the tube inside, put it in the heat block)
  4. Don't let it stand at room temperature, you can do it at 5 degrees (heat block)

Gel Extraction Protocol using QIAgen MinElute Kit:

  1. Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator. 
  2. Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV. 
  3. Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue. 
  4. Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 300mg per tube
  5. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul)
  6. Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
  7. Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
  8. Add 1 gel volume of isopropanol
  9. Add max of 800ul to MinElute column and centrifuge for 1 min (speed >= 10,000 G, RT)
  10. Discard flow-through and place column back in tube.
  11. If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
  12. Add 500 uL of buffer QG and spin column for 1 min and discard flow-through
  13. Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min
  14. Discard flow-through & centrifuge for 1 min
  15. Place column into clean Eppendorf tube
  16. Add 10ul Buffer EB (10 mM TrisCl,pH 8.5) or water to center of membrane
  17. Let stand at RT for 1 min
  18. Centrifuge for 1 min
  19. Measure the concentration using the UV spectrophotometer.
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