127Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Step 9. Extraction: Pellet the cell debris
Centrifuging enables you to separate materials of different densities. After the lysing process is complete, the
cells, along with cell debris in micelles and other aggregates, will be denser (have more mass per unit volume)
than smaller molecules that remain dissolved (like proteins and water molecules).
Microcentrifuges typically spin at 13,000 revolutions per minute (RPM) or more, which corresponds to an accel-
eration of more than 10,000 x g (italicized g is the force of gravity, not grams). This powerful force will cause
the denser molecules to fall to the bottom of the tube in a compact mass called a “pellet”. In this exercise, the
pellet will include micelles, macromolecules like lipids, bits of cell membrane, and even genomic DNA. Small
cellular components, such as proteins and DNA plasmids, may not be pelleted and can remain in the liquid.
Balancing the microcentrifuge. A microcentrifuge is meant for holding ‘microcentrifuge tubes’, which typi-
cally hold volumes between 0.5 mL and 2 mL. The Lysis Buffer, cells, and lysozyme in your tube of lysed cells
will amount to about 1.1 mL, so your tube is probably quite full.
Your centrifuge should always be balanced. This means you should place another tube of equal mass in the
open spot directly opposite from your Lysis Buffer tube. Inside the Plate Extract-it Kit, you will nd a Balancing
Tube which may already contain some water in it. If it does, note that while it will be close to the corresponding
mass you want, you should always double-check to make sure it is correct. To balance your tube, use a new
clean pipet to add tap water to the Balance Tube until the liquid level is similar. Use your small scale to weight
the tubes and conrm they weight the same.
The tubes should be within 5% mass of each other. For example, if Tube A = 2 g, then Tube B should be no more
than 2.1 g or less than 1.9 g because 5% = 0.05 x 2g = 0.1g. The closer the mass, the better. Make sure to close
the tubes back tightly after balancing them.
With your experiment’s lysis tube set opposite your balance tube, close the microcentrifuge lid(s) as per the
manufacturer’s instructions, tighten the lid, set the speed at the highest setting, and start the microcentrifuge.
Monitor to see if the microcentrifuge vibrates - if it does, stop it immediately! Note that the microcentrifuge will
hum and make some noises as part of its normal operation. A vibration means you either did not place your
tubes opposite of each another, or you have not adequately balanced the tubes.
Figure 5-8. Step 9. Pelleted cells in a microcentrifuge tube.
If this is your rst time using a microcentrifuge, set it up as per the manufacturer’s instructions. Then, start by spin-
ning the microcentrifuge without tubes in it. Try different speeds, starting slowly. As the rotor speeds up, there should
be no vibrations; it should be balanced. You should observe the same behavior when you add your balanced samples
into the microcentrifuge; they should not cause the microcentrifuge to vibrate.
If there is any vibration, immediately stop the microcentrifuge and re-balance your samples.
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