47. The capture and analysis settings for NTA must be consistent
across samples for comparability.
48. If the amount of cells is less than 10 g, cells can alternatively be
lysed by bead beating (next protocol in this chapter) or the use
of a probe sonicator. For probe sonication, prepare the cells in
breaking buffer according to Subheading 3.4, steps 1–4.
Probe sonicator settings should be 50% duty cycle, ON for
60 s, and OFF for 90 s. Each ON–OFF cycle should be
repeated at least 12 times, and sample should be kept on ice
at all times. Ice should be replaced periodically.
49. Each set of O-rings consists of a primary and secondary O-ring.
The primary O-ring is cylindrical and should be positioned
closest to the sample. The secondary O-ring has a flat side
and a concave side. Ensure that the secondary O-ring is
installed with the concave side facing the primary O-ring in
order to create a proper seal when pressure is applied.
50. The nut holding the plug in place should be tightened with a
wrench to prevent leaking. All other pieces should be fingertight only. Excessive tightening can damage the unit.
51. Care should be taken to hold onto the unit bottom, to prevent
sample loss due to the bottom falling off.
52. Care should be taken to point mouth of bottle away from the
face, as small air pockets inside the French press cell may cause
unpredictable, volcanic expulsion of eluent.
53. Sample may drip from the sample outlet tube when the vacuum
is released, so be sure to catch any drips with the sample
collection bottle.
54. Roughly 90% of the cells will be broken by this method. WCL
will appear as a field of blue debris (or green, depending on
counterstain used) with few pink bacilli. If cells are not broken,
pass back over the French press until sufficient cell disruption is
achieved.
55. The French press cell should be cleaned immediately after use
to prevent cell debris from drying and causing clogs in the
system.
56. If this method is used to lyse live cells, perform all steps with
open sample tubes in a BSC and inside a BSL-3 lab for pathogenic mycobacteria.
57. If bead beating is to be performed for RNA purification, cells
should be resuspended in Trizol instead of breaking buffer.
Trizol will also inactivate the cells.
58. Total volume of beads and cell suspension should not be more
than 1 mL if using 2 mL tube size. Ideal volume is one-third of
beads and two-thirds of sample.
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across samples for comparability.
48. If the amount of cells is less than 10 g, cells can alternatively be
lysed by bead beating (next protocol in this chapter) or the use
of a probe sonicator. For probe sonication, prepare the cells in
breaking buffer according to Subheading 3.4, steps 1–4.
Probe sonicator settings should be 50% duty cycle, ON for
60 s, and OFF for 90 s. Each ON–OFF cycle should be
repeated at least 12 times, and sample should be kept on ice
at all times. Ice should be replaced periodically.
49. Each set of O-rings consists of a primary and secondary O-ring.
The primary O-ring is cylindrical and should be positioned
closest to the sample. The secondary O-ring has a flat side
and a concave side. Ensure that the secondary O-ring is
installed with the concave side facing the primary O-ring in
order to create a proper seal when pressure is applied.
50. The nut holding the plug in place should be tightened with a
wrench to prevent leaking. All other pieces should be fingertight only. Excessive tightening can damage the unit.
51. Care should be taken to hold onto the unit bottom, to prevent
sample loss due to the bottom falling off.
52. Care should be taken to point mouth of bottle away from the
face, as small air pockets inside the French press cell may cause
unpredictable, volcanic expulsion of eluent.
53. Sample may drip from the sample outlet tube when the vacuum
is released, so be sure to catch any drips with the sample
collection bottle.
54. Roughly 90% of the cells will be broken by this method. WCL
will appear as a field of blue debris (or green, depending on
counterstain used) with few pink bacilli. If cells are not broken,
pass back over the French press until sufficient cell disruption is
achieved.
55. The French press cell should be cleaned immediately after use
to prevent cell debris from drying and causing clogs in the
system.
56. If this method is used to lyse live cells, perform all steps with
open sample tubes in a BSC and inside a BSL-3 lab for pathogenic mycobacteria.
57. If bead beating is to be performed for RNA purification, cells
should be resuspended in Trizol instead of breaking buffer.
Trizol will also inactivate the cells.
58. Total volume of beads and cell suspension should not be more
than 1 mL if using 2 mL tube size. Ideal volume is one-third of
beads and two-thirds of sample.
104
Megan Lucas et al.