Transcript Bild 1 - Uppsala University

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 information
why do you do this experiment?
Methods
all steps you do in the lab,
include all calculations made in preparing solutions
Results and conclusion
Overview
Day 1.
Mutation PCR reaction
Day 2.
DpnI digestion
Day 5.
Do small-scale expression test
Analysis: SDS-PAGE
Analytical agarose gel
Day 6.
Analysis: SDS-PAGE
Transformation to TOP10
Day 7.
Large scale fermentation
Day 3.
Day 8.
Purification (I)
Day 9.
Purification (II)
Pick 3 colonies, patch and
inoculate o/n cultures
Day 4.
Plasmid miniprep
Analytical PCR and
inoculate o/n cultures
Analysis: SDS-PAGE,
Protein concentration
Day 10. Activity test.
Where to store stuff
at room temperature:
DNA loading dye
10 mM Tris pH 8.5
5xSB
at 4C:
agar plates with bacteria on them (if you want to keep them)
cultures that you want to use as inoculum
-20C:
dNTP (keep on ice whenever out of freezer)
10xPfu buffer
10xTaq buffer
template
primers
DNA ladder
Amp
plasmids
samples 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 water
Rehydrate for 2 min (ie. leave on bench).
Tap the tube.
Working solution (20 µM):
20 µl 100 µM stock solution
80 µl autoclaved distilled water
Pipetting enzymes
in 50% glycerol  tip just below surface
look at the tip!!!