Lab safety Documentation, GLP Practical tips; primers and PCR
Jan 11, 2016
Lab safety
Documentation, GLP
Practical tips; primers and PCR
Lab safety
- wash your hands
- EtBr
- sterile technique
Lab safety
- Use Labcoats
- No food in the lab
- Common sense!
Lab safety
Documentation, GLP
Practical tips; primers and PCR
What can an experiment looks like?
Title should be descriptive!
Background informationwhy do you do this experiment?
Methods all steps you do in the lab, include all calculations made in preparing solutions
Results and conclusion
Day 1. Mutation PCR reaction
Day 2. DpnI digestion
Analytical agarose gel
Transformation to TOP10
Day 3. Pick 3 colonies, patch and
inoculate o/n cultures
Day 4. Plasmid miniprep
Analytical PCR and
inoculate o/n cultures
Overview
Day 5. Do small-scale expression test
Analysis: SDS-PAGE
Day 6. Analysis: SDS-PAGE
Day 7. Large scale fermentation
Day 8. Purification (I)
Day 9. Purification (II)
Analysis: SDS-PAGE,
Protein concentration
Day 10. Activity test.
Where to store stuff at room temperature:DNA loading dye10 mM Tris pH 8.55xSB at 4C:agar plates with bacteria on them (if you want to keep them)cultures that you want to use as inoculum -20C:dNTP (keep on ice whenever out of freezer)10xPfu buffer10xTaq buffertemplateprimersDNA ladderAmpplasmidssamples taken out for SDS-PAGE
Lab safety
Documentation, GLP
Practical tips; primers and PCR
Primers Materials:tube with dried primer. On the tube label, you can see how many nmoles there are inside. Experiment: Stock solution (100 µM): Spin the tube a few seconds.Open carefully and add 10 x (nmoles of primer) µl of autoclaved distilled waterRehydrate for 2 min (ie. leave on bench).Tap the tube. Working solution (20 µM): 20 µl 100 µM stock solution80 µl autoclaved distilled water
Pipetting enzymes
in 50% glycerol tip just below surfacelook at the tip!!!