Reagent handling

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Mol.biology // Next-generation sequencing



[править] Common reagents

  • maximal precautions against contamination when dealing with common stocks.
  • use new plastic pipettes or tips with filters for removing of solid reagents from the bottle.
  • if it is necessary to share reagent with other group newer give the whole bottle for some time. Prepare an aliquot and give it w/o return.
  • newer take reagents for experiment directly from the original bottle. Prepare an aliquot and use it. Use only clean (or specialized) bottle for aliquot.

[править] Reagent handling

  • all reagents should be marked by the date of arrival. Example is "A: 07/08", means "arrived in July 2008.
  • enzymes (kits, reagents) delivered on dry ice should be stored at -70°C until the first use.
  • be careful with solid reagents, a lot of them should be stored at +4°C or at -20°C, but are delivered on NT.

[править] Enzymes

  • do not put in ice, use Stratagene cool box
  • from freezer:
» spin down
» mix by fingertip
» spin down again
  • best tubes for enzymes: 0.5ml (require adapters for centrifuge and for Stratagene cool box):
» 0.2ml — unstable caps, too thin walls
» 1.5ml — 2-10µl tips would touch tube walls
  • storage: most of enzymes are completely stable at -70°C. Storage on -70°C is a compromise: there is some risk of inactivation during freezing, but on -20°C enzymes slowly (0.5-1 years) lose activity.

[править] Aliquoting

NB! The main problem with aliquots: it takes time and efforts to prepare aliquots, but the output is not obvious. It seems, that time and efforts were useless if nothing happened (contamination or reaction does not work).

  • keep your aliquote on your area (bench, freezer, fridge)
  • ideally, there should be NO tube where several users pipette from
  • better, when the stock of reagent is not completely common. Person, who predominantly uses some reagent, should supervise it

[править] Why reagents should be aliquoted?

  • when aliquoted, the same stock of a reagent might be used by several users
    • the reagent will be used faster, so will be substituted by a fresh stock earlier
    • if stock works well in hands of the first user, others may rely on their aliquots
  • in case of contamination suspicious aliquot might be thrown away, not the whole packing
  • often original tubes are inconvenient for handling:
    • a wide label completely hides the content of a tube
    • reagent is delivered in 1.7-2ml tube, but 2µl pipette should be used for dispensing (tip is too short and pipette will touch the walls of the tube)
  • moisture-sensitive reagents (e.g. fluorescent dyes) and enzymes should be aliquoted to minimize the number of freezing/thawing cycles
  • some enzymes are used rarely or should be used for a long period of time, it is better to store major part at -70°C, where the storage time might be much longer.

[править] How to aliquote

  • reasonable size of an aliquot is ~5-25 working portions (it is not a number of reactions, but a number of oppenings of tubes. So, if one person normally makes ~20 PCR reactions per one experiment, and another: ~2 reactions, than, aliquots should differ in 10 times)
  • for aliquoting use non-autoclaved tubes (LoBind — for valuable reagents) and tips with aerosol filters
  • make sure, that no contaminations will be introduced during aliqouting, use new batches of tips and tubes
  • do not aliquot in parallel with another work. Interrupt your experiments, aliquote (when possible) several reagents one after another and then come back to experiments
  • clean before start:
    • bench
    • markers
    • scissors
    • tube racks
    • centrifuge rotors (bench-top and nano-centrifuge)
    • -20°C box
    • vortex
    • metal block for melting
    • thermovortex
    • gloves (or use new)
  • moisture-sensitive reagents (SYBR's, DMSO, dry proteins, dry salts stored at -20°C) should be heated up to room temperature before opening (DMSO solutions are solid in freedge)

[править] Enzymes

  • enzyme solutions are viscous, they need more time for pipetting, spinning down or mixing.
  • try to avoid heating.

  1. prepare tubes for aliquots:
    • decide, how many aliquots you should make;
    • prepare labels for tubes and caps
    » enzyme name
    » activity and (optional) volume
    » arrival date[1]
    • prepare tubes (preferentially - low binding, in most cases 0.5ml, never use 0.2ml): sign tubes very shortly (and caps, if they are removable), stick labels, cool down tubes (preferentially to -20°C);
  2. prepare enzyme:
    • spin down enzyme (preferentially in cold room or cooled centrifuge);
    • mix by finger-typing;
    • spin down again;
    • check content of the tube:
    » if volume looks correct
    » if there is anything unusual in the tube: precipitate, color, etc.
  3. dispensing:
    • dispense aliquots into prepared tubes with a single filter-tip (or positive displacement tip)
    » do not use dispenser: too much material on the walls, massive dispenser will heat up enzyme
    » do not pipette too much volumes: too much material on the walls
    » start from 2-3 times pipetting back and forth to cool down the tip
    » push down pipette's plunger to the first position only to avoid bubbles
    • spin down the rest of enzyme and transfer it to the last tube;
  4. storage:
    • if tubes are to be stored at -70°C, snap freeze them in liquid nitrogen;
    • put on -70°C, write in "storage book";
  5. documentation:
    • put enzyme label and arrival date into "aliquots book"

[править] Buffers

  1. prepare tubes for aliquots:
    • decide, how many aliquots you make;
    • prepare labels for tubes and caps
    » buffer name
    » concentration and (optional) volume
    » arrival date[1]
    • prepare tubes (in contrast to enzymes, it is more convenient to dispense buffers into unsigned tubes, then close caps and sign them);
  2. prepare buffer:
    • melt buffer at room temperature (preferentially in metal block);
    • vortex and spin down;
    • (IMPORTANT!) check, that there is no precipitate (solubilize precipitate by vortexing; if necessary, use thermovortex 37°C for 5-10min);
  3. dispensing:
    • dispense aliquots into prepared tubes with a single filter-tip
    » push down pipette's plunger to the first position only to avoid bubbles
    • spin down the rest of the buffer and put it in the last tube;
  4. storage:
    • sign tubes: very shortly by marker (and caps, if they are removable);
    • stick labels on tubes and caps;
    • snap freeze tubes in liquid nitrogen, put them into the freezer (keep in mind, that they are too cold, do not put them nearby with enzymes).
  5. documentation:
    • put buffer label and arrival date into "aliquots book"

[править] Chemicals

  • small-weight aliquots: more practical to dispence roughly and write the weight on a tube
  • store H2O aliquots frozen

[править] If contamination happened (or reaction does not work)

  1. throw out:
    » all cheap reagents: H2O, Ethanol 100 & 70%, buffer aliquots, working aliquots of oligos, etc.
    » if there are some doubts: working aliquots of tips and tubes
  2. clean pipettes, forceps, desk, tip racks, tube boxes, etc.
  3. perform reaction in parallel with
    » all old (valuable) reagents
    » all new reagents (fresh aliquots)
    » (optional) all reagents from recovery box

  • usually (if problem happends once), it is not necessary to find a source of contamination. It is enough to remove it.

  • it is really nice, if a special "recovery box" with aliquots of all reagents is prepared for critical situation. "Recovery" aliquots should:
» be prepared when everything is fine
» contains all components from H2O to enzymes
» each reagent in several small (2-5 portions) aliquots
» stored frozen.

Critical situation may be really hard and difficult for the analysis. Three measures may help:

  • start new batches of buffers, enzymes, etc. when there are couple of old aliquots. If everything is fine, use old aliquots.
  • try to recognize problems as early as possible
  • prepare "recovery box" for important protocols.

  1. 1,0 1,1 to simplify tube signing use different arrival dates for different stocks
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