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Common techniques

Liquid handling

Pipetting

  • Make master mixes whenever possible to reduce measurement error, especially when working with small volumes. Add extra 2-4% to be sure you got enough for all your samples.
  • Enzymes in 50% glycerol: Do not submerge the pipette tip deep as too much enzyme will be aspirated due to glycerol's viscosity
  • Viscous liquids (e.g., glycerol, mineral oil):
    • Pre-wet the tip by pipetting up and down before taking the final amount
    • Can also fully press it to aspirate, then release to the soft press, and discard the rest
  • Volatile liquids (e.g., ethanol): Hold the pipette at an angle after aspiring the final amount and work fast as the liquid quickly starts to drip.
  • Always watch that all components enter and exit the pipette tip.

Mixing / vortexing

  • Mixing up and down with a pipette: repeat 10 times
  • Vortex and centrifuge tubes before opening, except for sensitive compounds like enzymes.
  • Enzymes in solutions usually can be vortexed briefly.

Centrifuging

  • Balance your tubes roughly by volume. It need not be perfect: for instance, when balancing spin columns, people often use a different kind of a column as a counterweight (if the correct one is not available, of course).
  • For larger centrifuges, balancing is done by weight of the entire thing that you put in the centrifuge, including, for instance, a paper label.
  • Do not overfill your tube in such a way that once it is tilted in the centrifuge and there was no cap, the liquid would spill.

Cooling

  • Keep your reagents on ice unless specifically mentioned otherwise.
  • Aluminum and water-based Eppendorf racks warm up at about 3-4 deg/min. Thus, if you take something from -20C, in about half an hour your sample will reach 0 deg and in an hour it will be room temperature.

Concentrations and weights

  • Dilutions: Final conc. = Stock conc. x Input vol. / Final vol.
  • Concentration in M = Mass (g) / (Molar weight (g/mol) x Volume (L))
  • 1 uM = 1 pmol/mL
  • DNA weight to concentration:
    • Concentration in nM = 1623 x Concentration in ng/uL / Size in bp
  • When preparing solutions, first dilute components in a smaller amount of a solvent than the final amount would require, then add to reach the final volume. This is done because the volume may change when solid components dissolve.

Working with RNA:

  • Work at a RNAse-free workstation / laminar hood.
    • Clean surfaces with RNAse removal spray (e.g., RNAsaZap). But note that residues of RNAseZap might damage RNA, so rinse with RNAse-free water.
    • Sterilize with UV after usage.
    • Autoclaving does not completely eliminate RNAse contamination.
  • Protective clothing:
    • Clean lab coat.
    • Fresh gloves (change them after making contact with anything outside of the RNAse-free zone).
    • Face mask (to avoid airborne contamination).
  • Samples:
    • Store reagents closed and open them only when needed (to avoid airborne contamination).
    • Store samples with ribonuclease inhibitors to maintain RNA integrity.