User:Jarle Pahr/Checklists

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'''Miniprep:'''
'''Miniprep:'''
*Centrifuge is available
*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.
*After measuring DNA concentration, record statistics (culture volume used, OD600, yield, etc.) for use in procedure optimization.

Revision as of 08:40, 21 May 2013

Checklists for lab procedures:


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).


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
  • 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
  • Freezer box is marked.

Electrophoresis:

  • GelDoc 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).
  • Wipe down lab bench with ethanol.
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