User:Jarle Pahr/Checklists

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Checklists for lab procedures:

Checklists are useless if they are not used. Print the checklists and consult them when doing experiments.

Start of day:

  • Wipe down lab bench with ethanol.


General experiment checklist:

  • Prepare all materials the day before. Ensure that all necessary items are available/reserved and ready to use, including flasks/bottles. Tubes, pipette tips, etc.
  • If possible, perform a dry run of the experiment beforehand to. This may include pipetting of water to simulate samples, if pipetting operations might need planning.

Fluorescence experiments:

  • All necessary strains are inoculated, including controls.
  • Appropriate controls are included. As a rule, a consitutive expression positive control and a negative control with as similiar genetic background as feasible should be included. Ideally, the negative control should include a wild-type strain. If feasible without too much work, include two separate control of each type. For example, for a negative control, use two different non-fluorescent strains.
  • When performing measurements in cultures inoculated from an overnight culture, measurements should be performed on the overnight culture first to confirm expected signal patterns of positive and negative controls.
  • "Gain" setting is set to automatic or manual, as appropriate.
  • Instrument is reserved.
  • Transfer and layout of samples on microplate is planned. Take into account the output format to reduce the number of spreadsheet operations that must be performed to collate measurements. When performing performing biological replicates, use the same layout for all the plates. Describe the sample layout in the raw data file.
  • Instrument software: All wells to be measured are included in the program.
  • To increase reproducibility, for any given transformant, preferably use cultures inoculated from glycerol stock, either directly or pick colonies on an agar plate spread from that glycerol stock. Preferably, do not use colonies directly from agar plates used in transformation, or if you do, use glycerol stock derived from that colony only for further experiments. (Of course, if behaviour of the culture is not as expected, consider re-transforming/re-growing, but this should be noted).
  • Microplate cover is on/off as appropriate, and the correct option in the software is chosen.
  • In the output file or in another easily identifiable file, note the experimental procedure/protocol used. and the identity of samples in the wells.

Before starting measurements, check systematically that the physical and software setup is correct. This includes:

  • Plate type and cover on/off
  • Temperature if doing an incubation/kinetic experiment
  • Shaking
  • Wells to be measured
  • Excitation and emission wavelengths.
  • Measurement types (fluorescence, absorbance)
  • Check that instrument gain value is set.


After experiment: Perform a quick sanity check of data.


Transformation:

  • Supercompetent cells are available (remove from freezer 20 min before use, to avoid waiting for cells to thaw).
  • Enough plates, with right antibiotic, are available. Remove from cold storage, place in incubator for pre-heating.
  • Waterbath for heat shock is at right temperature (42 C).
  • Ethanol for sterilization of glass spreader is available. (use absoulute ethanol).

Before heat shock: Place plates in incubator for pre-heating.


Restriction digest:

  • Needed restriction enzymes are available.
  • Thaw buffer
  • Waterbath at 37 C is available. (or otherwise, thermomixer, or otherwise, shaking incubator)


Ligation:

  • Ligation buffer is available.
  • Ligase is available.
  • Correct ligase is used (T4 DNA ligase)
  • If possible include positive control to make sure the ligase is functional.
  • If possible, include negative control of bacbone only/insert only, as appropriate.


Pipetting:

  • Release plunger slowly to avoid air bubbles forming at the tip.
  • When pipetting small volumes (enzyme reactions, etc.), confirm both that the right volume is drawn into and expelled from the pipette.

Preparation of agar plates:

  • Magnetic stirrers are available
  • Antibiotic is available. Thaw antibiotic.


Preparing supercompetent cells:

  • Centrifuge is cooled. Chill tube holders of the correct size by placing them in the centrifuge in advance.
  • Spectrophotometer and cuvettes for measuring OD are available
  • When using spectrophotometer: Correct wavelength is selected (600 nm)
  • For snap freezing: Dry ice or liquid nitrogen is available.
  • Freezer box is available
  • Sterilize tubes for cell aliquots are available.
  • Freezer box is marked.

Electrophoresis:

  • Imaging system is operational
  • After imaging: Save image as image file. Copy file(s) to personal area.
  • Remove gel from imaging apparatus.
  • Copy image to document, write observations.


Gel extraction:

  • The correct band is cut.

Measuring OD:

  • Instrument is set to correct wavelength (typically 600 nm)
  • Cultures are re-placed in incubator immediately after sampling.
  • Enough liquid in cuvette
  • Cuvette is inserted in correct orientation

Miniprep:

  • Centrifuge is available
  • Before centrifuging, measure OD600 is possible, for records on relation between culture properties and DNA yield.
  • After measuring DNA concentration, record statistics (culture volume used, OD600, yield, etc.) for use in procedure optimization.


Use of instruments, general:

  • After end of run/experiment, copy data to personal area.
  • Print paper of copy of raw or processed data, place in ring binder or lab journal.


Inoculating cultures:

  • A suitable incubator and tube rack / holder for the culture(s) is free.

Receiving plasmid samples for use:

  • As soon as possible, verify the sequence (entirely, or critical parts only) for yourself
  • Make a new glycerol stock for your personal use.

End of day:

  • No samples are left on bench (that shouldn't be there).
  • Update sample records
  • Wipe down lab bench with ethanol.
  • All inoculated cultures and plates are in their correct place for incubation or storage.
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