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HighMwGenomicDna
HighMwGenomicDna

High Molecular Weight Genomic DNA

Mechanical shearing forces involved in typical protocols for GenomicDnaIsolation preclude isolation of DNA fragments much larger than 50 kbp in size. For higher molecular weight DNA, it is necessary to isolate the DNA from cells while supported by a porous agarose matrix. The isolated DNA can be subsequently digested by restriction enzymes while still embedding in the agarose and then directly loaded on a gel. DNA fragments of several megabases and larger can be isolated by this method.

There are two different ways to imbed the genomic DNA in agarose: in plugs / tubes, or in agarose microbeads. Microbeads have a very high surface-area to volume ratio which greatly improves the diffusion-mediated access of restriction enzymes to the DNA embedded in the agarose, but they are trickier to make and more difficult to manipulate. In general, the use of agarose tubes (aka "worms") suffices.

For both tubes and microbeads:

  1. melt certified nuclease-free low-melting-temperature (LMT) agarose at 1.2% weight/volume in PBS (phosphate-buffered saline without Ca+2 or Mg+2, tissue culture grade) at 70C, place the melted gel solution in a waterbath or oven at 42C and let temperature equilibrate
    • it is convenient to use LMT agarose that melts at ~65C and gels around ~23C
  2. with adherent cells, treat with trypsin/EDTA to acheive a cell suspension
    • stop the trypsin/EDTA action by addition of 2 volumes of serum-containing medium
    • if starting with whole blood, first do the LeukocyteIsolation protocol
  3. count cells
  4. spin down cells, resuspend in PBS-EDTA to rinse
  5. spin down cells again and resuspend in PBS-EDTA to 3x107 cells / ml, put in a waterbath or heatblock at 37C
    • be sure to take into account the volume of the packed cell pellet when resuspending the cells. the cells have some volume to them and if this is not accounted for, the final gel concentration will be lower than 0.8% and hard to work with, and the cell concentration will be lower than 1x107 / ml
  6. add two volumes of melted 1.2% LMT agarose solution to the cell suspension. mix well to make a suspension of 1x107 cells / ml in 1% LMT agarose
    • the melted agarose is viscous and difficult to pipette accurately. Cut the end off a 1000 μl pipette tip to get a wider opening and draw the melted agarose up and down into the pipette tip several times to warm and pre-coat the pipette tip before dispensing
    • if making agarose microbeads, mix the cells and agarose in a glass culture tube pre-warmed to 42C (see the microbead-specific protocol below)
  7. optional: for every 1 ml of agarose/cell mixture, add 20 μl (about one drop) of a 2.5% suspension of colored polystyrene 0.2 μm microspheres (eg Polysciences Inc.) and mix thoroughly
    • adding the colored microspheres can greatly aid subsequent manipulations, especially for agarose microbeads which are otherwise transparent to nearly the point of invisibility

For agarose worms:

  1. draw the cell/gel suspension into a 1 ml syringe. immediately place the syringe on ice to solidify the agarose before the cells have a chance to settle
  2. after the gel has solidified, cut the end off the syringe. Extrude the gel carefully into an 8 ml flat-bottomed tube
  3. add 5 ml of digestion buffer, invert gently to get the agarose worm suspended and incubate overnight at 50C with gentle agitation
    • this 50C incubation can be for more than one day if you prefer. probably overnight will suffice
  4. pour off the digestion buffer. transfer the worm to a 50 ml tube with about 40 ml TE and agitate gently at room temperature for 10 minutes
    • Caution: the worms are slippery and will try to escape onto the floor, or into the sink. don't let them do that
  5. repeat the above step one more time
  6. decant the TE and transfer the worm to a new 8 ml flat-bottomed tube. add 6 ml TE. add saturated PMSF in isopropanol to a final concentration of 0.1%. agitate gently at room temperature for 60 minutes
    • WARNING: PMSF is very toxic. handle with care.
    • PMSF is a serine-protease inhibitor to inactivate the proteinase K from the digestion buffer. This will prevent residual proteinase K from destroying restriction enzymes used to digest the genomic DNA
    • PMSF is quickly inactived in aqueous solution and needs to be added fresh from the saturated isopropanol stock immediately before use, every time
  7. pour off the solution. add fresh TE without PMSF. gently agitate at room temperature for 60 minutes
  8. repeat the above step one more time
  9. chill TE-glycerol to 4C or on ice
  10. pour off the TE and add back chilled TE-glycerol. gently agitate at 4C for about 30 minutes
  11. repeat the above step with fresh chilled TE-glycerol
    • if the TE-glycerol is warm (RT or above), the worm will shrink in diameter, making it loose and difficult to cut when subsequently returned to a syringe. Accoring to NEB, the worm has fully equilibrated when it sinks in the chilled TE-glycerol solution
    • experience indicates that shrinkage at room temperature is minimal, so this may suffice. use 4C if you are worried, or have a problem at room temperature
  12. transfer the worm back to a new syringe and store at -20C

For agarose microbeads:

  1. if not done already, transfer the cell/agarose suspension to a glass, round-bottomed culture tube pre-warmed to 42C, 18mm outer diameter x 150mm length
    • it is critically important to use a glass tube
  2. add 2 volumes of pre-warmed 42C paraffin oil to the cell/agarose suspension in the culture tube
    • example: 1 ml of cells + 1 ml of 1% agarose + 4 ml of paraffin oil
  3. vortex vigorously by placing the culture tube on a vortexer and intermittently lifting the tube on and off the vortexer until the oil and agarose are thoroughly emulsified
    • lifting the tube off and onto the vortexer repeatedly helps make the emulsion fine by mixing the solution up and down the inner sides of the culture tube
    • the emulsion should look milky and homogeneous
    • if the oil/cells/agarose mixture is too cool, the agarose will begin to solidify before the emulsion can be made. when this happens, you will get amorphous agarose blobs from which cells can escape rather than discrete agarose beads. start the entire procedure again if this happens
  4. immediately continuously swirl the culture tube in an ice-water bath until the agarose in the emulsion has solidified
    • this takes about a minute
  5. add an equal volume of room temperature 0.1EX solution to the solidified emulsion, thoroughly mix by pipetting up and down repeatedly, transfer to a 15 ml conical centrifuge tube
    • subsequent steps can be done at room temperature. take care not to heat the microbeads above the melting temperature of the low melting agarose
  6. centrifuge for 5 minutes at 2500 g in a swinging-bucket clinical centrifuge
    • centrifuging at typical forces for pelleting tissue culture cells such as 200 g will not be sufficient to pellet the microbeads effectively
  7. aspirate off the top layer of frothy paraffin oil, and most of the underlying liquid, being careful not to aspirate the microbeads
    • the microbeads will be difficult to see if colored microspheres were not added but will show a subtle phase transition if the tube is held up to the light
  8. add back 10 ml fresh 0.1EX and pipette up and down to resuspend the microbeads. examine the microbeads under a phase-contrast microscope. the beads should be from 0.1 to 0.3 mm in diameter. dark, oval nuclei should be visible embedded in the beads
    • the plasma membrane is not visible since it is immediately dissolved by the Triton-X100 in the 0.1EX solution
  9. transfer the resuspended microbeads to a new 15 ml conical centrifuge tube and respin for 5 minutes at 2500 g.
  10. pour off and discard the liquid supernatant
    • the beads should be tightly enough packed in the bottom of the tube that they will not be lost if the supernatant is carefully decanted
  11. resuspend the microbeads in 5 ml of digestion buffer, incubate overnight with gentle agitation at 50C
  12. pellet the microbeads by centrifuging 5 minutes at 2500 g. pour off and discard the digestion buffer. resuspend the beads in 10 ml of 0.1EX. Examine the beads again under a phase-contrast microscope (10x objective, 10x eyepiece). You should see ringed fainter "holes" in the beads where the cells used to be before they were digested.
  13. Add saturated isopropanol PMSF solution to a final concentration of 0.1% (v/v). Mix well and incubate at room temperature for at least one hour.
    • WARNING: PMSF is very toxic. handle with care.
    • PMSF is a serine-protease inhibitor to inactivate the proteinase K from the digestion buffer. This will prevent residual proteinase K from destroying restriction enzymes used to digest the genomic DNA
    • PMSF is quickly inactived in aqueous solution and needs to be added fresh from the saturated isopropanol stock immediately before use, every time
  14. repeat the previous step one more time to change in fresh 0.1EX with fresh PMSF
  15. centrifuge 5 minutes at 2500 g. pour off and discard the 0.1EX with PMSF. estimate the volume of the microbead pellet and resuspend in an equal volume of 0.1EX without PMSF. store the microbeads at 4C


SOLUTIONS

digestion buffer
500 mM EDTA, pH 8.0
1% sarcosyl (sodium lauroyl sarcosinate)
0.5 mg / ml proteinase K → added immediately before use

PBS-EDTA
PBS is phosphate buffered saline without Ca+2 or Mg+2
add 1/50th volume of a 500 mM EDTA, pH 8.0 solution to make PBS with approximately 10 mM EDTA final concentration

TE
10 mM Tris pH 8.0
1 mM EDTA pH 8.0

TE-glycerol
10 mM Tris pH 8.0
1 mM EDTA pH 8.0
50% glycerol

0.1EX
100 mM EDTA pH 8.0
0.01% (v/v) nuclease-free Triton-X100

saturated PMSF in isopropanol
add isopropanol to PMSF (phenylmethylsulphonylfluoride) crystals to make a saturated solution. Some PMSF crystals should remain undissolved in the bottom of the container. Store the saturated PMSF isopropanol solution indefinitely at room temperature


REFERENCES


COMMENTS

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