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Mol.biology // Next-generation sequencing



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[править] Oligonucleotide storage

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

[править] Oligonucleotide handling

NB! work with oligonucleotides stocks in a pre-PCR area

number of oligos:


  1. determine nmol/OD extinction coefficients for all oligos;
  2. calculate how much EB it is necessary to add to get

    namequant. [nmol]sizenmol/OD260OD260conc. [µM]initialcorrection

  3. solubilize oligos (take care not to loose the dry pellet!):
    • centrifuge lyophilized oligos for 3min at a maximum speed
    • add EB and vortex for 30 sec
    • incubate in a thermoshaker at 37°C for ~15min (or rotate: {0.5-1h at normal temperature} or {overnight at +4°C})
    • vortex for 30 sec and spin down
    • check, that there is no undissolved material in the tube
  4. measure oligonucleotide concentration:
    NB! for accurate measurement oligonucleotide concentration should be: in spectr. cuvette ~1-2µM; for Nanodrop: ~10-20µM
    • prepare tubes with 1xSTE (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);
    • determine the concentration;
  5. (optional) check oligonucleotide quality on denaturing PAGE;
  6. add EB to shift oligonucleotide concentration to a convenient value:
  7. vortex 20 sec and spin down;
  8. prepare working aliquot;
  9. snap freeze in liquid nitrogen, store at -20°C.

Test-electrophoresis of oligonucleotides
test-electrophoresis should check:

  • integrity of oligos: correspondence to expected size and absence of smaller-size bands
  • accuracy of quantitation: correct concentration

To solve the first task load oligos on a gel according to their sizes, e.g. from shorter to longer, it is easier to notice 1nt difference, if oligos are on neighbor lanes. To check concentration measurement, load the same optical amount on each lane. Oligonucleotide shadows should look the same in this case.

sample volume:

Oligonucleotides2x formamide
Orange G
loading mix
nameconc. [µM]sizenmol/OD260volume

  1. calculate amount of oligonucleotides for loading;
  2. prepare gel and samples;
  3. run the gel:
    • assemble the gel chamber, fill in the running buffer (preheated to ~60°C in a microwave);
    • pre-electrophoresis 30 V/cm, 15min at 50°C; (in a thermostat);
    • heat samples at 95°C for 5 min, then put immediately in ice-bath;
    • wash wells with a syringe, load samples, run oligos into gel: 30 V/cm 5min;
    • put chamber at 50°C, run until Orange G dye reaches the bottom of the gel;
  4. stop elecrophoresis, open gel and cut out right-upper corner of the gel;
  5. vizualization:
    • wrap gel with Saran wrap and put it on a sheet of white office paper;
    • check image under UV 260nm and make a photo.

[править] Solutions

[править] STE, 10x

store at +4°C

Tris Cl, pH7.50.1M1M0.1ml0.5ml
H2O mQ680µl3.4ml

  • sterilize by autoclaving or filtration

[править] EB, 10x

store at +4°C

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

  • sterilize by autoclaving or filtration

[править] Notes

  • typical time for operations:
» dissolving: ~1h
» test-electrophoresis: 1.5-2h w/o gel preparation

  • measurement of oligonucleotide concentration: spectrophotometer only. Fluorescent measurement should be calibratd for each particular oligonucleotide

nmole/OD260 = 106 / ε260 = 106 / { 2(∑1N-1εNearest-Neighbor) - ∑2N-1εIndividual + ∑1NεModification} ~ 106 / (2*(s-1)*10162.5[1] - (s-2)*10750[2]);

  1. mean nearest-neighbor molar extinction coefficient
  2. mean individual molar extinction coefficient
Источник — «http://zbio.net/wiki/Oligonucleotides»

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