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Oligonucleotides

Oligonucleotide handling
     Dissolving
     Test-electrophoresis
     Storage
Oligo purification in PAGE
Notes
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Oligonucleotide handling

NB! working with oligonucleotides stocks is a pre-PCR operation

number of oligos:

Dissolving (~1h w/o electrophoresis)

  1. calculate how much EB it is necessary to add to reach
    OligonucleotidesMeasurementEB
    namequant. [nmol]sizenmol/OD260OD260conc. [µM]initialcorrection


  2. solubilize oligos (do not loose a dry pellet!):
    • centrifuge liophylized oligos for 3 min at maximum speed;
    • add EB and vortex for 30 sec;
    • incubate in thermoshaker (37°C) for ~15min (or rotate: {0.5-1h at NT} or {ON at +4°C});
    • vortex for 30 sec and spin down (check, that there is no undissolved material in the tube);
  3. measure oligonucleotide concentration:
    NB! for accurate measurement oligonucleotide concntration in spectr. cuvette should be ~1-2µM; concentration for Nanodrop: ~10-20µM.
    • prepare tubes with 1x STE (for all oligos + one control):
    • add oligonucleotide solution to each tube (EB for control):
    • measure optical density on spectrophotometer (not necessary to wash cuvette between samples);
    • calculate concentration;
  4. (optional) check oligonucleotides on denaturation PAGE;
  5. add EB to shift oligonucleotide concentration to convenient value:
  6. vortex 20 sec & spin down;
  7. prepare working aliquote;
  8. snap freeze in liquid nitrogen, store at -20°C;



Test-electrophoresis of oligonucleotides (1.5-2h w/o gel preparation)

    test-electrophoresis should check:
    • integrity of oligos: expected size and lack of smaller-size bands;
    • accuracy of quantitation: correct concentration
    to solve the first task put oligos on electrophoresis according to their sizes. For the second — load the same optical amount on each lane. Oligonucleotide shadows should look the same in this case.

  1. calculate amount of oligonucleotides:
    quantity:
    sample volume:
    Oligonucleotides2x formamide
    Orange G
    loading mix
    nameconc. [µM]sizenmol/OD260volume

  2. prepare samples and denaturating PAGE;
  3. run gel:
    • pre-electrophoresis 30 V/cm, 15min at 50°C;
    • heat samples at 95°C for 5 min, then put immediately in ice-bath;
    • wash wells, load samples, run oligos into gel: 30 V/cm 5min;
    • put chamber at 50°C, run until Orange G reaches the bottom of the gel;
  4. stop elecrophoresis, open gel, cut out right-upper corner of the gel;
  5. vizualization:
    • wrap gel into the Saran wrap & put it on office paper;
    • check image under UV 260nm & make photo;



Oligonucleotide storage

    conditiontime
    liophylized, +4°C or -20°Cyears
    solution at -20°Cyears
    solution at +4°Cweeks



Oligonucleotide purification from PAGE (~40min before, and ~3h after elution)

    Prepare in advance shearing assembly: 0.5ml tube without cap and with two holes in the bottom inserted into 2ml colection tube. To make holes use 28G needle or red-hot preparative needle, holes should be in "cap-side" of the bottom.

  1. put gel on Saran-wrap;
  2. localize band by UV shadowing:
    • put wrapped gel on office paper;
    • illuminate gel by UV 260nm in the dark (use as less as possible UV light — close part of the UV-lamp window by piece of plastic);
  3. cut out band and transfer it into 0.5ml shearing tube;
  4. add 50µl of 2M LiClO4 into 0.5ml shearing tube;
  5. spin down gel through the shearing holes: 14krpm 0.5 min;
  6. discard 0.5ml shearing tube;
  7. add 300µl of 2M LiClO4 (up to 350µl) into 2ml collection tube;
  8. elute oligonucleotides by diffusion in thermoshaker at 60°C for 2h (or rotate overnight at NT);
  9. remove gel pieces by centrifugation through 0.45µm spin filter;
  10. add 1ml of acetone to eluate, mix and incubate 1-2h (or ON) at -20°C;
  11. centrifuge 14krpm 30min at 4°C;
  12. wash pellet with 200µl of ice-cold acetone (NB! do not loose the pellet);
  13. dry out pellet 15 min at NT;
  14. add 15µl EB;
  15. measure concentration on spectrophotometer or Nanodrop;
  16. snap freeze in liquid nitrogen, store at -20°C;


    Notes

  • Electrophoresis: it is possible to add more, than equal volume of 2x GL-buffer;


  • Oligonucleotide purification:
    • this protocol works well for purification of 1-20nmol of oligos. Normal yield is 30-50%;
    • in our hands acetone precipitation results in cleaner oligos, than reverse-phase chromatography (better results in LDR-TaqMan reaction);
    • loading capacity of denaturating PAGE is ~2µg/mm2;
    • UV light damages DNA. To reduce UV light exposure close part of the lamp window;
    • if part of the gel remains in 0.5ml shearing tube, add 50µl of 2M LiClO4 and spin down once more;
    • it is worth to make photos of gel before and after band cutting.





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