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- Don't chill the cell to -20oC or your sample will freeze when you put it in the block. If you need to chill a cell fast, put it in an ice bucket in the cold room.
Collect your equipment:
- 1- Cell cylinder
- 2- Screw valve
- 3- Piston
- 4- Cylinder cap
- 5- Spigot
Prepare the equipment:
- Check the condition of the O-rings on the piston and cylinder bottom before starting your extraction
- - If they are shredded, sticky or warped you should change them
- - There are 2 types of O-ring, flat and rounded; the rounded O-ring should be positioned on the outside
- Check the condition of the nylon ball housed in the screw valve
- - The ball must be rounded to effectively adjust pressure
- - Flattened or squared off balls should be changed out
- - Use a razor blade to pry the malformed ball out of the housing and place a new ball in the opening
- Refridgerate setup at 4°C until thoroughly cold
You must have someone show you how to operate the press itself if you have never used it, but here is an overview of the press controls:
- 1- Pressure gauge
- 2- Pressure dial (increase/decrease psi)
- 3- Ratio selector (up/down)
- 4- Platform/riser (for micro cells)
- 5- Guide plate (swings to secure the fully withdrawn piston)
Pressing a sample
Turn on the machine and set working pressure:
- Toggle machine on
- Use the pressure dial to increase to a MAX of 1000 psi (for micro cell cylinders)
Prepare your sample:
- Invert the assembled cylinder, remove the cap, and pull the piston back until you see the grooves indicating max volume
- Load up to ~3-4 ml of sample into the core of the cylinder
- Make sure the screw valve is slightly open (otherwise the air trapped in the core will not have a release and you will have a hard time getting the cap to seat properly)
- Fit the cap onto the cylinder, making sure the O-rings are snug inside the core
- - Here is where you run the risk of losing sample, either out the spigot (if you force the cap on too hard and the valve isn't cracked), or out of the core (if you overfilled it); go slow! There is also no shame in pipetting up any accidentally ejected sample from the countertop and adding it to the next run!
- If you put fewer than 3 mls of sample in the core, while the cylinder is still inverted you can depress the piston a little bit (you will get a feel for this as you use the press more)and release air in the core through the spigot
- As gracefully as you can, flip the fully assembled cylinder, with piston pulled back, into the upright position
- Tighten the screw valve, but only "finger tight"!
- - DO NOT wrench it closed -- over-tightening will cause your nylon ball to flatten or shear, and can result in annoying repairs to remove pieces stuck in the cylinder outlets
- - While the press is in operation, you are going to manually adjust the pressure inside the cylinder using this valve, and it's a very gentle adjustment...turns will only be a fraction of an inch to increase or decrease pressure!
Put the cylinder onto the platform:
- Swing the guide plate into place
- Adjust the cylinder orientation so that you can operate the screw valve and also collect the sample as it exits the spigot
- Be especially careful that the arms on the top of the piston will not be crushed by the screws protruding from the guide plate!
Start the press:
- Check to make sure the pressure gauge indicates 1000 psi
- Using a bit of muscle, pull the ratio selector level from "down" to "medium" position
- (I think if you don't have the riser black you would select "high"?)
- The press is going to slooooowwwwllllly elevate the platform upwards, AND IT SEEMS WAY TOO FAST THE FIRST TIME IT IS HAPPENING, but I promise you have time to pick up your collection tube in your right hand, and poise your pressure-adjusting left hand on the screw valve
- - It's a good time to double check that your valve is closed (but not too tightly!!)
- - The valve twisted towards you tightens and increases pressure
- - The valve twisted away from you loosens and decreases the pressure
- When the piston reaches the top of the machine, it will plunge into the core until it hits the sample
- - Since the valve is closed (but not too tightly!), pressure inside the system will increase to 1000 psi
- Here is where things get real; you now need to find the sweet spot in valve tightening and loosening that maintains constant 1000 psi while also allowing the release of pressed sample
- - Very carefully, and very slowly, twist the valve away from you while simultaneously monitoring the pressure gauge
- - When the valve has been released just enough for the sample to evacuate the core, it will begin to burble out of the spigot
- - It's very important to maintain 1000 psi to evenly process your cells!
- - Carefully back off the pressure and increase the pressure as needed to keep 1000 psi and release pressed cells
- - When you get to the end of the sample in the core, and the piston seems to be fully depressed, all the cell debris that hasn't come out the spigot will have built up
- - Fully release the valve pressure, and all the remaining junk will splat out all at once
- When piston is fully depressed, and all your processed cells have been collected, turn the ratio selector back to "down"
- - You do not need to wait for the platform to be fully lowered before you remove the cylinder
- - It's a great time to refill the core with remaining sample, or load what you collected back into the core if you need to process your sample twice
- Repeat as necessary
Completing your run
- When you are completely finished with the machine, you MUST release the pressure before switching it off!
- -Turn the pressure dial down to zero, then flip the on/off switch
- Wash the cylinder and parts with ddH20 followed by 70% EtOH
- Return the cylinder to the cold room
- Tidy up the shared work area!
- - Wipe down counters and machine surfaces
- - Pack up your pipette tips/serilogical pipettes, ice buckets, tubes, etc., etc.
- - Campfire rules = leave the station in better shape than you found it!