Oligonucleotide purification from PAGE

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 Problem list:

  • LiClO4, 2M


Mol.biology // Next-generation sequencing

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Содержание


[править] Preparing shearing assembly

  1. make two holes ~1mm in diameter in the bottom of 0.5ml tube:
    » use 28G needle or red-hot preparative needle
    » holes should be in "cap-side" of the bottom
  2. cut out a cap of 0.5ml tube;
  3. put 0.5ml tube into a 2ml collection tube with a cup



[править] PAAG electrophoresis of oligonucleotides

Loading gel capacity of denaturing PAGE is ~2µg/mm2:

  • gel thickness [mm]:
  • width of the well [mm]:
  • maximum amount of oligonucleotide [µg]:



[править] Oligonucleotide purification

  1. cut out band[1]:
    • put gel on Saran-wrap
    • localize band by UV shadowing:
    » put gel (on Saran-wrap) on a sheet of white office paper[2]
    » illuminate gel by 260nm UV in the darkness (use as less as possible UV light[3])
    • cut out band and transfer it into 0.5ml tube of the shearing assembly
  2. elute oligonucleotides:
    • add 50µl of 2M LiClO4 into 0.5ml shearing tube
    • spin down the gel through shearing holes: 14krpm 0.5 min[4]
    • discard 0.5ml shearing tube
    • add 2M LiClO4 up to 350µl to 2ml collection tube
    • elute oligonucleotides by diffusion in a thermoshaker at 60°C for 2h (or rotate overnight at normal temperature)
  3. precipitate oligonucleotides:
    • remove gel pieces by centrifugation through 0.45µm spin filter inserted in a 1.5ml tube
    • add 1ml of acetone to eluate, mix and incubate 1-2h (or overnight) at -20°C
    • centrifuge 14krpm 30min at +4°C
    • wash the pellet with 200µl of ice-cold acetone (NB! do not loose the pellet)
    • dry out the pellet 15 min at normal temperature
    • dissolve in 15µl EB
  4. store:



[править] Solutions

[править] EB, 10x

store at +4°C
pH8.5

conc.stock1ml5ml
Tris Cl, pH8.50.1M1M0.1ml0.5ml
H2O mQ900µl4.5ml

  • sterilize by autoclaving or filtration



[править] LiClO4, 2M

Solutions/LiClO4, 2M

[править] Notes

  • time: ~30min before, and ~3h after elution from gel
  • the protocol works well for purification of 1-20nmol of 20-120b oligos. Normal yield is 30-50%
  • we routinely use 100x105x1.5mm3 PA gels for oligos purification
  • PAAG should be "well-polymerized[5]" otherwise unfiltrable mass would form after addition of acetone
  • we routinely repurify
» long (>40nt) oligonucleotides
» adaptors for cloning
» shorter oligos if their quality (length and absence of shorter sequences) is important for downstream applications
  • it is not necessary to repurify primers for analytical PCR
  • in our hands acetone precipitation results in cleaner oligos, than reverse-phase chromatography (better results in LDR-TaqMan reaction)




  1. it is worth to make photos of gel before and after band cutting. It is possible to use normal electronic camera.
  2. office paper have the same sensitivity as a chromatographic plate. Additional advantage: it is possible to use a new paper sheet for each oligo.
  3. UV light damages DNA. To reduce UV light exposure close part of the UV-lamp window by piece of paper or plastic
  4. if some gel remains in 0.5ml shearing tube, add 50µl of 2M LiClO4 and spin down once more
  5. degassed, fresh APS, etc.


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