So it didn't work
No growth on LB/amp plates
There are many different possibilities as to why you had no growth:
1. Make sure you heated the plates at 37˚C for the full 30 minutes.
2. Make sure you actually placed sample on your plates.
3. Make sure the plate is not dehydrating overnight. Our incu-shaker tends to dehydrate plates if they are not poured thick enough or if the petri dish shape allows too much air flow. You can tell that a plate is dehydrated when it looks like this:
In the corner of the plate you can see that there is a crack in the agar.
You can see that there is a dip in the agar or uneven agar right in the middle which indicates that it was dehydrated.
4. If all of that is not the case, it could be possible that there is too much ampicillin in the mixture and nothing is able to grow. In this case you would need a whole new set of plates. Ampicillin tends to go bad and we tend to continue using it just at a higher concentration. So when you do get a fresh batch make sure you lower the concentration back down or else nothing will grow.
Overexpression is not working or small pellet size
What is most important is that all the ingredients in your media are not expired or if they are new you are adding the correct amount. For media check that the Yeast Extract is not old. It should not be clumpy and sticky it should be in a powder form. Potassium phosphate dibasic is super important it must be good in order for this to work.
IPTG must be good! If it isnt good you have to add more or just end the overexpression it is very important for transcription and for cells to form. We fixed this by aliquoting the IPTG in water the minute we get it rather than allowing it to stay in powder form.
Arabinose must be added and must be good without arabinose cells will not grow!
AMPICILLIN!!! Make sure your ampicillin is good. If it is not good we tend to increase the amount until we finish the old bottle. Make sure that when you do get a fresh batch of ampicillin that you correct back to the normal amount you should be adding. Otherwise nothing will grow and your protein will be unfolded.
Protein Purification is not working
A lot of things can go wrong with the FPLC and/or your set-up:
1a: Unusually low overexpression: You NEED Protease Inhibitor in your whole cell and lysate to prevent the cell recycling machinery from breaking down your protein once the cell is lysed.
1b: My lysate won't spin down the cell debris cleanly / I can't clarify my lysate. You may not have included DNAseI in your whole cell incubation which means the DNA unspooled and is making everything gooey. DNA can contact both the lipid bylayers of organelles and is charged enough to be soluble in water so it will make separating these two components really hard. On the upside, you can add this late and it will still work, just give it another 10-15 minutes to clip the DNA into smaller pieces.
2. If your protein prep went flawlessly and nothing is missing, but you still are getting overpressure alerts then: clean the FPLC with 10% methanol for at least 0.25 L, and possibly replace the column you are using with a new one. Make sure you are carefully clarifying your lysate and not loading any insoluble cell debris into the pump or the column in the future.
3. If the UV is very bumpy then: There is likely a bubble in the detector. Flush with copious amounts of solvent (magic water or 10% methanol) until it flattens out.
4: You know you need to deep clean the system when: Baseline noise increases. Deep cleaning never hurts anything so don't be hesitant.
5. Your protein is eluting off too early: Make sure you resuspend your cells in low imidazole buffer, and that lines A and B are in low/high imidazole buffers, respectively. Additionally, ensure that the column is well washed with low imidazole buffer before and after every run.
6. If you see that it is not fractionating centered on each tube: manually check that the arm is fully seated over the tubes and that the fraction collector is centering the tubes under it. When moving the fraction collector bucket, always pull the gasket in the back away from the bucket so as not to strip the teeth when adjusting the tube position.
7. If the system seems to be running incorrectly as in not asking you to switch the injection valves at correct timing: watch it like a hawk.
8. If your conductivity(red line) is very bumpy or moving a lot: remake solutions or flush the system.
SDS-PAGE Gel is not working
Not much can go wrong but:
1. If the screen says E1 or Error 1 check that the 1/10x Tris/Glycine/SDS Buffer Solution is at a high enough level for the amount of gels you are running. Also make sure that there are no leaks in your gasket and that the Buffer Dam is facing the right direction of the gasket.
2. If after dying your gel you see that there are many lines and it seems as though it is contaminated, but you know it is not: you could have forgotten to take off the green strip at the bottom of the gel. If you do this it will continue running, but it will be very bumpy and not usuable data.
3. If you pulled out the comb very crooked, get a needle or syringe filled with the buffer and manually move and fill the wells so that they are straight.
4. If you are having trouble loading Whole Cell or lysate, make sure that you did the right dilution (EG usually does a 2/30x), make sure that you heated the samples at 95˚C for the full 2 minutes.
5. If your gel is very dehydrated or dropped in size and is beginning to roll up, rehydrate with Magic water. (If anything looks funky about your gel just put it in magic water and it will fix almost anything.)
Growth in autoclaved broth
This can only be due to one thing:
1. You did not completely seal every single hole on the glass material you are autoclaving. A lot of the flasks we use have spouts for vacuum filtration those holes MUST be covered especially when the media is TB overexpression broth.
Vacuum Filtration is taking too long
1. Check to see that all the connections are solid, if any air is getting out it will take much longer.
2. Make sure to prime the filter with a little squirt of water first.
3. If none of the above help, make sure you are using a good vacuum with good oil in it.
4. If really none of the above are true, your filter must be old and you should say thank you to it and good bye. Note that high concentrations of glycerol (ex: 25% in ITC buffer) make it harder on the filter and vacuum so be patient. Only throw away a filter if it is filtering liquid through VERY slowly i.e. dropwise.
The incubator/shaker won't cool/power-up
It won't cool below room temp: Be sure you turned on the right (power and heat) switch on the front right side. If you are cooling, you'll need the left front switch (cooling below room temp) ALSO flipped on. If you aren't cooling below 21oC, leave that off for longer fuse life.
It won't even turn on! There is a known issue with fuses blowing on our shaker. Let Dr. Hoffmann or Dr. Swig know and they will replace the fuse (this will take about 20 minutes total, you can either wait with your samples or use another incubator/shaker in the meantime.)
Overexpression does not work
Overexpression does not occur due to contaminated lab water/residue left in magic water, must utilize cleaner water in order to continue on. Options for cleaner water include: HPLC grade water (make sure it hasn't been sitting in the bottles for years) or distilled water from ex: the Water Store in Ventura.
If you suspect contaminated water, you'll need to remake the following with clean water:
- LB Agar plates
- Plasmid preps (depending on when they were prepared, they are eluted off the column into magic water)
- LB or TB broth
- All the purification buffers
- As an advanced move, pre-rinse containers with clean water if you have been cleaning them with the contaminated water.