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yeast
E. coli
plant tissue
muscle & organ tissue
cultured cells
Before using the Bullet Blender™
please note
General guidelines
• order of addition in the tube: tissue, beads, then
buffer.
• tubes: Use
polypropylene tubes. Microcentrifuge tubes: a 1.5mL or 1.7mL tube may perform
better than a 2.0mL microcentrifuge tube because the
bevel on the bottom of the tube is steeper, allowing
more mixing. Use quality brand tubes such as
Axygen. 50mL tubes: use BD Falcon or Krackelerbrand
tubes; other tubes which are slightly different in size
will note work well.
•
volume of sample: When the tissue is
overloaded, there is not enough airspace in the tube to allow for efficient mixing
of the sample with the grinding agent. The movement of
the liquid around the walls of the tube is important and
without an airspace, you will not get effective
homogenization.
- small tissue samples: As the
tissue amount becomes smaller, that ratio will differ due
to the limitations of handling of the small volumes.
Cutting the tissue into smaller pieces will generally
yield better results.
- with microtubes:
We
recommend less than 300mg (that is 0.3g of tissue) per
microtube in the Bullet Blender™. You must leave at least 500
microliters of airspace in a
microcentrifuge tube to get effective homogenization.
- with 50 mL tubes: For similar reasons, do
not fill the 50 mL tubes with more than 15 mL total of
buffer, tissue, and beads.
• volume of beads:
- glass beads: 1 volume of tissue:1 volume
of grinding media: 2 volumes of buffer.
-
denser beads: 1 mass of tissue: 1 to 1-1/2 mass of
grinding media: 2 mass of buffer. (When there are a
lot of the denser beads in the tube, they tend to settle
at the bottom and weigh the tube down from shaking
vigorously, so best results are obtained when using equal
mass instead of equal volume of beads to tissue.)
• choice of beads:
Check our
bead selection guide for recommended grinding agent
for each application and the
FAQs
section below the guide.
• volume of buffer: usually two volumes of buffer to
one volume/mass of tissue
• Considerations for RNA isolation:
- Use fresh
biological material and nuclease free labware, reagents,
and beads. For treatment of beads, see the
FAQs on our
beads webpage.
- Follow the
homogenization protocol for your particular sample using
Trizol® as a homogenization
buffer.
- Remove the tube and visually inspect the homogenate. If
there are unacceptably large pieces of tissue remaining,
run for another 2 minutes.
- Note that the amount of Trizol® used to homogenize the cells
is less than what the manufacturer recommends for RNA
isolation. After homogenization, add the rest of the
volume of Trizol® specified by the manufacturer's protocol.
For example, use 0.2mL Trizol® in the Bullet Blender™ to
homogenize 100mg tissue. After the homogenization is
complete, add 0.8mL Trizol® to the tube (to meet the
manufacturer's specification of 1 mL). Next, close
the tube and invert it several times (5-10) to ensure
complete mixing. Proceed with the manufacturer's protocol
to isolate the RNA.
Typical disruption protocol
1.
Isolate tissue from animal. Process as quickly as possible
into small pieces (~50mg
each, about 1mm x 5mm x 5mm).
•
Thinner slices are better for homogenization than large
“chunks”.
2.
Place tissue slices into a microcentrifuge tube on ice.
•
Use between 50-300mg of tissue with microcentrifuge tubes.
•
Less dense, less fibrous tissues may be easier to disrupt,
so more may be used.
3.
Add cold buffer to tissue.
•
Generally, two volumes of buffer for each volume of tissue
are desired to
maintain high concentrations of material to be analyzed in
the lysate.
•
Volumes ranging from 100µL to 1000µL may be used depending
on the
toughness of your tissue and the particular application
you have in mind.
•
The contact time between tissue and adjuvant grinding
materials (e.g. glass
beads, stainless steel balls, etc.) is important to
disruption efficiency. There will
be
some volume above which you will notice a significant
decrease in efficiency,
but these results will vary with the toughness of the
tissue, size of tissue pieces,
volume of buffer, and volume of the grinding material.
4.
Add grinding material (if desired).
•
Use a volume of grinding material similar to the volume of
tissue.
•
Use grinding material of similar diameter to the tissues
to be disrupted.
5.
Place tube in the Bullet Blender™
•
Set the desired power and time.
•
Close the lid and secure the lid latch.
•
Push the start button.
6.
After the Bullet Blender™ has stopped, remove the tubes
and proceed in your
application.
Generally, disruption and lysis are performed
simultaneously for whole cell extracts.
Any inhibitors required to maintain the quality of the
desired cellular material (i.e.
Protease inhibitors, Rnase inhibitors, etc.) should be
added to the lysis/disruption buffer
before the tissue is exposed to the buffer.
To
assess the amount of lysis in your application, use a Lowry
or Bradford assay. When
starting, it is helpful to run a small pilot experiment to
determine the best parameters for
your tissue processing. The protocol supplied here is a
good place to start, but given
that biological materials vary greatly, you will need to
determine the ideal quantities for
your experiments.
For protein or
DNA applications, beads should be autoclaved or acid
washed, but do not require
additional preparation. However, when RNA extraction
applications are desired extra care must be taken to avoid
decomposition of RNA from exogenous RNase activity. To
ensure that beads are not the source of RNase activity, we
recommend washing the beads with RNase Zap®
(Ambion, Inc.) or RNase Away®
(Molecular BioProducts), then rinsing with nuclease free
water, followed by autoclaving.
Below we list some settings from user feedback.
Please send us your parameters and we will add them to
this list.
Disrupting Yeast Cells: 3 minutes at a speed of 8
using 0.5 mm glass beads.
Test results in mg/mL of lysates, performed in duplicates:
0 min - 0; 1 min - 1.58, 1.32; 2 min - 1.94, 1.7;
3 min - 2.54 ; 2.33
E. coli
and Yeast Genomic DNA
Prep: Adapted from the Rather Rapid
Genomic Prep from the Fred Hutchinson Cancer Research
Center.
Centrifuge 5mL overnight saturated yeast culture to pellet
the cells.
Resuspend pelleted yeast cells in 500 uL water.
Transfer to 1.5mL microcentrifuge tubes, then spin briefly
(5000g, 15sec).
Aspirate supernatant.
Resuspend the pellet in 0.2mL buffer (2% Triton-X, 10% SDS,
100mM NaCl, 10mM Tris, 1mM EDTA)
Add 0.2mL glass beads to each tube (omit this step for E.
coli).
Add 0.2mL phenol:chloroform:isoamyl alcohol (25:24:1)
Place tubes in the Bullet Blender, running at a speed of 8
for 3 minutes.
Add 0.2mL TE, spin at max speed for 5 minutes.
Prepare a new microcentrifuge tube with 1mL 100% ethanol.
Transfer the aqueous layer (~0.4mL) from the yeast lysate
to the tube containing the ethanol.
Spin the crude genomic DNA at maximum speed for 5 minutes
(4°C).
Pour the supernatant off of the crude DNA pellet, then
resuspend in 0.4mL TE.
Add 10 microliters 5M ammonium acetate, then 1mL ethanol
(100%).
Centrifuge the mixture at maximum speed for 5
minutes (4°C).
Pour of the supernatant, then dry the pellet and resuspend
in 50 microliters of sterile water.
E. coli
and
Yeast genomic DNA prep using 50 mL tubes:
When using the Bullet Blender™ 50
for larger E.coli and yeast cultures,
overnight cultures from 50mL to
150mL may be used. A 50mL culture will be
most convenient, as it will fit in
the 50mL conical centrifuge tube all at
once. Larger cultures will require
multiple centrifugation steps
to combine the cell pellet in one
tube. Scale up reagents according to
the culture size (i.e. in steps
using 2-6mL, use 2mL for 50mL culture,
4mL for 100mL culture and 6mL for
150mL culture)
After harvesting the cell pellet,
resuspend in 2-6mL lysis buffer (2% Triton-X,
10% SDS, 100mM NaCl, 10mM Tris, 1mM
EDTA).
Add a volume of
phenol:chloroform:isoamyl alcohol (25:24:1) equal to the
volume
of lysis buffer (i.e. 2-6mL).
Add 2-6mL 0.1 mm diameter glass
beads to each tube (omit this step for E. coli).
Place tubes in the Bullet Blender™
50, running at a speed of 8 for 3 minutes.
Add 2-6mL TE, centrifuge at 6000rpm
speed for 15 minutes.
Prepare a new centrifuge tube with
10-30mL 100% ethanol.
Transfer the aqueous layer
(~4-12mL) from the lysate to the tube containing the
ethanol.
Spin the crude genomic DNA at
maximum speed for 5 minutes (4°C).
Pour the supernatant off of the
crude DNA pellet, then resuspend in 4-12mL TE.
Add 100-300µL ammonium
acetate (5M), then 10-30mL ethanol (100%). Mix.
Centrifuge the mixture at maximum
speed for 5 minutes (4°C).
Pour off the supernatant, then dry
the pellet and resuspend in 250-750µL sterile water.
E. coli
and Yeast Protein Prep:
Centrifuge 5mL
overnight saturated yeast culture to pellet the cells.
Resuspend pelleted
yeast cells in 500 uL water.
Transfer to 1.5mL
microcentrifuge tubes, then spin briefly (500g, 15sec).
Aspirate supernatant.
Prepare one portion
of lysis buffer/protease inhibitor mixture for each of
the samples.
0.3mL lysis
buffer (2% Triton-X, 10% SDS, 100mM NaCl, 10mM Tris, 1mM
EDTA)
0.3mL 25%
glycerol
0.02mL
phenylmethylsulfonylfluoride (25mM in ethanol)
other
protease inhibitors as desired
Add 0.6mL lysis buffer/protease
inhibitor mixture to each sample.
Add 0.2mL glass beads to each tube (omit this step
for E. coli).
Place tubes in the
Bullet Blender, running at a speed of 10 for 5 minutes
(4°C).
Remove the tubes from
the Bullet Blender, centrifuge at maximum speed for 10
minutes (4°C).
Pipette the clarified
protein lysate from the cellular debris into a new
microcentrifuge tube.
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Plant Tissue:
Leaves: Place
4 ZrO beads, 2mm diameter, in a 2.0 mL microtube, along
with 250 uL of buffer. Snap 1/2 cap of a fresh green
leaf. Then add another 250 uL of buffer. (If
you add all the buffer before the leaf sample, the leaf
will float). Run at speed 8 for 3 minutes.
A. thaliana leaf tissues and seedlings: For tissue
homogenization prior to protein and genomic DNA
extractions. Prior to tissue collection place 3 or 4
stainless steel beads, around 1.5mm diameter, into the microcentrifuge tube.
Then
collect the tissue and snap freeze the tube in liquid
nitrogen. Once frozen, place tubes into the bullet
blender and pulse the samples 3 or 4 times to disrupt the
tissue. After adding extraction buffer (whatever amount
the protocol calls for, usually around 450-500uL)
homogenize in the blender at speed 8 for 2 or 3 minutes.
This extraction step has yielded high quality results in
beta-GUS fluorimetric assays and has made PCR-screening
transgenic lines a much smoother process.
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Tissue
and Organs:
Chicken muscle:
0.2 g of tissue
cut
into several pieces, equal volume of 0.5 mm diameter
zirconium silicate or zirconium oxide beads, 0.5 mL
buffer, speed 8 for 3
minutes.
Beef muscle: 200 mg of tissue
cut
into several pieces with 300 mg of 0.5mm zirconium
silicate or zirconium oxide beads. Run 2 minutes at speed 8
with 0.4 mL buffer.
Test results in mg/mL of lysates, performed in duplicates
with 0.3 g of 0.5mm glass beads.:
0 min - 2.25; 1 min - 3.25, 4.3; 2 min - 4.92, 5.14;
3 min - 5.31, 5.33
Liver:
200 mg of beef liver cut into several pieces with equal
volume of 0.5mm zirconium silicate or zirconium oxide
beads and 0.4 mL buffer. 2
minutes at speed 8.
Test results in mg/mL of lysates, performed in duplicates
using 0.3 g of 0.5mm glass beads:
0 min - 2.62; 1 min - 6.8, 8.11; 2 min - 9.42,
10.46; 3 min - 10.54, 10.11
Mouse jejunum and stomach tissue:
The tissue was frozen in liquid nitrogen (no fixative), we
used a setting of 8 for 2 minutes and put 200 µl of 0.5 mm
glass beads in an Eppendorf tube and 600 µl of Qiagen
buffer.
Trachea: 200 mg of
tissue, i.e. entire rat trachea. 0.2 g of 1.6mm
stainless steel balls.
400 microliters PBS (or 1:2 PBS/acetonitrile mixture).
Hair: 25 mg of
hair, 1.0 mL of buffer, 0.1 mL of glass beads, 10 min at
95C, then place in Bullet Blender for 3 min at speed 8.
Spin 2 min at 14K
RPM.
Lung (using 50 mL tubes):
0.9g rat lung (whole lobe), 2.5mL PBS, 5.0 mL
acetonitrile, 6.7g 3/16 in. (4.8mm) stainless balls.
6 min speed 8 in Bullet Blender™ Blue 50, centrifuge 500 rpm 5
min. Also successful: 0.7g rat lung (whole lobe)
tissue, 10.0 mL PBS, 6.7g 3/16 in. (4.8 mm) diameter
stainless steel balls.
Aorta: Load 3
stainless steel beads (1.6 mm dia) in each microcentrifuge
tube. Add 25 - 50 uL buffer (your choice, depending
on your downstream application). Place dissected
tissue into the buffer, then place the tube into the
Bullet Blender™. Set the time to 4 minutes and the
speed to 8.
Brain:
Load 100 uL of glass beads (0.5 mm dia) in each
microcentrifuge tube. Add 100 - 200 uL buffer
(your choice, depending on your downstream application).
Place about 100 mg of dissected tissue into the buffer, then place the
tube into the Bullet Blender™. Set the time to 3
minutes and the speed to 6.
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HeLa Cells: HeLa cells
lyse very effectively using 2x107cells/mL
in lysis buffer without beads, with the Bullet Blender running for 2 min
at a setting of 5 in a cold room (4°C), to yield a lysate containing ~5mg/mL
protein. HeLa cells
were trypsinized, washed with PBS and resuspended in a
mild lysis buffer (0.5% NP-40, 100mM KCL, 10mM HEPES, 5mM
MgCl2) at
a density of 2x107
cells/mL. The measurements were made using
the BioRad Quick Start
Bradford Dye Reagent with bovine serum albumin as the
protein standard and absorbance at 595 nm measured on the
NanoDrop spectrophotometer.
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