Rate Zonal Centrifugation

From OpenWetWare
Jump to: navigation, search

Linear Sucrose Gradients for Ribosome Recovery

Getting Started

Turn on the Beckman Ultracentrifuge
The arrow on the key should be facing the swinging bucket icon, and the power switch is on the right side.
(1) Set the centrifuge temperature to 4°C.
(2) Turn on vacuum. Watch the display to make sure it goes from a solid < 200 to a blinking < 20.
(3) Set the acceleration and deceleration speed to max.

Bring the SW-41 buckets up for inspection and put them in order
(1) Check inside the buckets for dried sucrose/ other debris.
(2) After 3-4 runs, remove the rubber O-Ring from the bucket with a 10 μl pipette tip
(3) If O-Ring is dry, apply a thin layer of Beckman vacuum grease silicone with gloved fingers
(4) If O-ring is compromised, discard and replace with freshly lubed O-Ring
(5) Take SW-41 buckets back down to cold room

Preparing the Gradients

To harvest polysomes, use a 10-40% sucrose gradient.
Each sucrose solution is prepared with HT-10 (20 mM Hepes-Tris, 100 mM K-Glutamate, 10.5 mM MgOAc, 0.1 mM EDTA, pH = 7.4 @ 4C).

(1) Weigh out the appropriate amount of sucrose to make your gradients (plan for an excess of 2-3 mLs to rinse the syringe).
(2) Use Beckman Polyallomer 14 x 89 mm (9/16 x 3 ½) tubes (opaque/ thin) if you plan on fractioning from the bottom. Use Seton tubes if you're fractioning from the top (Gradient Station)

  • Use the marking tool to indicate the fill line when dispensing the 10% sucrose. The lower platform is for the long caps and the higher platform is for the short caps.
  • Gradients should be prepared in the cold room for immediate use. (This includes dissolution of the sucrose in HT-10)

(3) Rinse the 10 ml syringe/ medium sized cannula (20#) with di H2O and subsequently, the 10% sucrose solution.
(4) Aspirate ~6 mLs of 10% sucrose.
(5) Dispense the 10% into the bottom of the Polyallomer tube to the fill line.

  • Rinse the syringe with 40% sucrose before the next step.

(6) Slowly dispense the 40% sucrose solution below the 10% solution until the 10% layer reaches the top of the tube.

  • While dispensing, be sure to keep the tip of the cannula just blow the 10% layer so that it displaces the smallest volume possible.
  • Following the initial drop with your cannula minimizes disturbance to the 10% layer.

(7) Insert the appropriate size silicone caps. The short cap and long cap displace enough sucrose to load 200 μl and 500 μl, respectively.

  • Try to eliminate any air bubbles before mixing the gradients.

(8) Turn on the Gradient Master and select the GMST option at the boot screen.
(9) Choose SW-41 -> ShortSucrose 10-40% w/v program.
(10) Grab your tubes, place them in the appropriate carousel, and select the "Run" option.
(11) After the program is finished, take the gradients back to the cold room or fridge and let them chill out.

Preparing to Spin

(1) To clear your lysate, spin it at 16,000 RCF for 5-10 minutes in the cold room.
(2) Slowly remove the silicone caps from the Polyallomer tube and load the supernatant on top of the gradient.

  • There shouldn't be much space left at the top of your centrifuge tube (seton suggests <= 2 mm), as this will lead to the plastic collapsing at high rotor speeds.

(3) Place the Polyallomer tubes into the buckets. They should float down in an ethereal fashion.
(4) Turn caps counterclockwise to engage the threads, then tighten them clockwise until resistance against the O-Ring is felt.
(5)Bring the SW-41 rotor to the ultracentrifuge with one hand holding the stem (depicted by the dotted arrow) and the head (depicted by solid line) resting on the shoulder.
(6) Carry the bucket rack from the cold room to the ultracentrifuge.
(7) Carefully load the buckets onto the rotor making sure that they swing freely
(8) Turn off vacuum and wait for the pump to stop (audible)
(9) Gently open door to chamber and carefully place rotor onto the spindle. Once on the spindle, turn rotor left and right until it clicks into place.

  • Do not rush while loading up the rotor and placing it in the centrifuge. This will lead to mistakes and also cause sloshing of the gradients.

(10) Select the speed and time for which you'd like to spin. (35,000 RPM/ 151,000 RCF for 2.5 hours for polysomes)
(11) Make sure the vacuum begins to drop and pay attention to any vibration perceived at harmonic intervals ( 1/4,1/3,1/2,2/3,3/4 of the final speed)

Fractionation and Visualization

Disclaimer: This guide is NOT a substitute for reading the manual provided by Bio-Comp.

  • Turn the Gradient Station, Bio-Rad LP machine, and Bio-Rad fraction collector on.
  • Start LP Data View program on the computron.
  • Blank the UV detector if you wish.
  • Load the fraction collector with the appropriate number of tubes

Gradient Station Instructions
1) Change the pipette tip that leads to the fraction collector.
1) Screw the piston tip onto the piston until resistance is felt. Do not over tighten.
3) Liberally lube the piston tip with silicone vacuum grease before each run. The position that the tip touches the tube is most important, don't be shy.
2) Select the "Frac" option at the boot screen.
3) Select the "Rins" option and turn the dial to 12 o'clock. Make sure "1 Fc" is chosen if continuous fractionation is desired. Exit.
4) Reset the knob to full CCW position.
6) Carefully snap your centrifuge tube into the lid of the tube holder.
7) Fill the tube holder to the sharpie mark on the acrylic window if you plan to image the gradient.
8) Insert the tube into the holder and turn the lid CCW to lock everything in place.
9) Use the control knob to bring the piston tip to the top of the gradient. Go slowly, this is easy to muck up.

  • The window allows visualization of the tip breaching the tube.

10) Press "Rset" 4) From the "Frac-Rins-Stup" screen: select Frac -> Frac -> Sngl. Set Speed = 0.1 mm/s, Dist = 91.00 mm, and Numb = 1 cycle.
10) Select the timed option on the fraction collector and choose 0.55 minutes/ fraction. This is roughly equal to 0.5 mL fractions.
10) Start collecting data by clicking the "Record" option in LP Data View.
11) Press Start on the "Frac-Rset-Up-Mrec" Screen
12) Export data using the /file/export ; select uv data only and 0.5s.