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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:

1. You NEED both DNAse and Protease Inhibitor in your whole cell and lysate.

--- Without DNAse, your lysate will be too stringy and viscuous making it dificult to work with. Also the FPLC will get clogged, give you many overpressure alerts, and you will ruin the column on the FPLC. 

--- Without Protease Inhibitor, your lysate will completely strip the Ni column of any Nickle. You can tell this happened when the color of the column is no longer the right shade of blue or is not blue at all. In addition, the proteases within the DesD will ruin the structure of your protein by cutting it up into smaller pieces.

2. If your protein prep went flawlessly and nothing is missing, but you still are getting overpressure alerts then:

--- Check that the superloop is VERY cleaned, possibly take it apart completely, give it a good scrub, and regrease the plungers.

--- Check that there are no kinks in any of the lines. If there are, we have a hose cutter in the tool box for the FPLC.

--- Check that none of the screws you placed for the hoses are actually caps.

--- Otherwise you probably have a clogged line somewhere and you need to deep clean the system.

--- Possible protein degradation because of inactive protease inhibitor or not enough protease inhibitor.

3. If the UV is very bumpy then:

--- Begin by doing a manual run of Magic water throughout the entire system. Watch this clean to see if anything comes of the column.

--- Check that there is good quality and the right amount of TCEP in your buffers. Without this you will see a bumpy UV at the end of your run.

--- If that still does not work, you can backflush the UV (Ask for assistance from Dr. Hoffmann)

--- If that does not work, you can run detergent through the entire system. NOTE: you must flush the system with magic water afterwards or else your next protein purification will be ruined.

--- Possible protein degradation because of inactive protease inhibitor or not enough protease inhibitor.

4: You know you need to deep clean the system when:

--- There is not equal amounts of fractionation throughout all the tubes. Something is clogged :(

--- The superloop is not loading all your sample.

--- You keep getting overpressure alerts.

5. Your protein is eluting off too early:

--- Try lowering the %B concentration to 1 or 2 % elution.

--- Try to program to start fractionation earlier (your protein may be contaminated so this is not ideal)

--- Reconsider the purification buffers. As in too much imidazole in your Buffer A.

6. If you see that it is not fractionating centered on each tube:

--- Make sure the black holder is pushed all the way into place so that it is aligned with the microcentrifuge tubes.

--- Make sure that the green hose is pushed all the way down into the holder so that you can see it under the black holder.

7. If the system seems to be running incorrectly as in not asking you to switch the injection valves at correct timing:

--- Make sure you turned on/off the system in the correct order. This is very important for computer-to-machine communication. If you did in fact do this in the incorrect order you will have to restart the entire system and hope that your protein is not lost. (Ask for assistance from Dr. Hoffmann)

8. If your conductivity(red line) is very bumpy or moving a lot:

--- Check that your buffers have the right concentration of salt.

--- Check that you used the right buffer for your protein prep.

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 whole on the glass material you are autoclaving. A lot of the flasks we use have spouts for vacuum filtration those wholes MUST be closed 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.