User:Jarle Pahr/Checklists: Difference between revisions
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*If possible, include negative control of bacbone only/insert only, as appropriate. | *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:''' | '''Preparation of agar plates:''' | ||
Line 73: | Line 77: | ||
'''End of day:''' | '''End of day:''' | ||
*No samples are left on bench (that shouldn't be there). | *No samples are left on bench (that shouldn't be there). | ||
*Wipe down lab bench with ethanol. |
Revision as of 03:54, 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
- 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.