APPLICATION NOTE #201413

Total Genomic DNA and Protein Extraction
from Escherichia coli K12 Cells on the Bead Mill 4
Shari Garrett

Introduction
Escherichia coli (E. coli) is frequently used
as a model organism in microbiology
and molecular biology studies. E.coli
was one of the first organisms to have its
genome completely sequenced which
has allowed it to be a major contributor
to recombinant DNA technology. The
process of gene cloning in E.coli involves
a series of steps including the isolation oftarget DNA or protein. Analyte extraction
is typically done through sonication
or bead milling. Sonication is the mostpopular technique for extracting proteins.
With the sonication process, the cells are
lysed via cavitation produced by a highfrequency oscillating probe to disrupt
cellular membranes releasing the internal
cellular components. There are a numberof drawbacks to the use of sonicators
including excessive heat generation,
variations in yield and sonicators are only
able to process one sample per cycle.
Ultrasonic homogenizers also shear DNA
making them unsuitable for many DNA
applications.
As an alternative to ultrasonichomogenizers, bead mills are higher
throughput homogenizers that are able toprocess multiple samples at once throughthe rapid oscillation of tubes containing
high speed projectiles that impact anddissociate cells. The efficiency of thebead milling process allows it to quickly
extract cellular compounds from cultured
cells such as E. coli while maintaining theirmolecular integrity. The Fisher ScientificBead Mill 4 (BM4) is designed as apersonal use bead mill to facilitate
efficient lysis of tissues and cells.

The BM4 is capable of homogenizing up
to four 0.5 mL, 1.5 mL, 2mL or 7 mL
samples using bead media to assist in
sample disruption process.
Here, we demonstrate the extraction ofDNA and protein from E. coli K12 cells
using the Bead Mill 4. Extraction efficiency
and analyte integrity was evaluated.

Materials & Methods
Equipment•

Bead Mill 4 (Cat No. 15340164)

Sample Temperature
0.7 g of 0.5 mm glass beads was added tofive 2 mL screw cap polypropylene tubes
with 1 mL of DDH2O water. Each tube wasprocessed in the Bead Mill 4 at allavailable speeds (1-5) for 30, 45, 60 and 75
seconds and the intratube temperature
was recorded.
DNA Extraction and Separation
5 mL of E. coli K12 stock culture (CarolinaBiological) was divided into three 1.6 mL
aliquots in 2 ml polypropylene reinforcedtubes. Each aliquot was centrifuged at
11,000 x g for 10 minutes to pellet cells. Thesupernatant was removed and each cellpellet was re-suspended in 750 µl of lysis
solution provided from the ZR Fungal/
Bacterial DNA Miniprep kit (ZYMOResearch).

Each cell suspension was added to2 mL tubes containing 0.5 mm glass beads(Cat No. 15340152 ). The suspensions werethen homogenized in the Bead Mill 4 (Cat#15340164) at speeds of 3, 4 and 5 for 45
seconds. After processing, the lysates weretransferred to clean 2 mL tubes andcentrifuged at 10,000 x g for 1 minute topellet any cell debris. DNA extraction wascarried out per the manufacturer’s
instructions using the ZYMO Research
Miniprep kit. 1 µl of the DNA elution wasquantified on a NanoDrop
Spectrophotomer (Thermo Fisher) todetermine DNA yields.
10 ul of each sample was mixed with 10ulof sample buffer (Bio-Rad). The DNA wasseparated on a 5% TBE agarose gel (BioRad) at 150 V for 45 minutes and stained inethidium bromide for 15 minutes. The gel
was washed for 10 minutes in DD H2O andvisualized on a GelDoc EZ System (Bio-Rad).
Protein Extraction and Separation
A 15 mL nutrient broth culture of E.coli
K12 was grown up overnight at room
temperature. The cells were harvested bycentrifuging the culture at 3,000 x g for
10 min. The cells were washed twice andresuspended in 1 ml Tris-HCL (pH 7.6). The
suspension was added to a 2 mL tube
containing 0.7 g of 0.5 mm glass beads andprocessed on the Bead Mill 4 for 60 seconds
at a speed of 5. The lysate was then cooledon ice for 5 minutes then centrifuged at
11,000 x g for 5 minutes to pellet cell debris.
The supernatant was removed and cooledon ice for another 5 minutes. 1 µl of thesupernatant was analyzed at 280 nm on aNanoDrop spectrophotometer todetermine protein

Results
In this study we determined the Bead Mill
4’s capability of extracting DNA andproteins from E. coli K12 cells. The
isolation of these compounds from E. colicells is critical because E. coli is used
extensively as a host for introductionof DNA and protein sequences and is a
critical scientific research tool.
As a first step, the intratube sample
temperature was monitored over time andas a function of BM4 speed setting todetermine the extent of temperature
increase due to the homogenization
process. The purpose of this study wasto evaluate temperature as a functionof processing speed and time. It wasobserved to even at the highest speed
setting and processing time of 75
seconds the intratube temperature did
not exceed 34°C (Figure 1). By precoolingthe tubes to 4°C prior to processing,extended processing can be performedat the highest speed of five without
temperatures exceeding 20°C for up to 75
seconds. This makes the BM4 ideal for
extraction of heat sensitive compounds
such as RNA from very tough tissues.
DNA and protein yields were quantified
by spectrophotometry (Table 1). Genomic
DNA yields averaged 23 ng/µl and wereindependent of speed setting. Genomic
DNA was fully liberated from the E. coli
cells at speed exceed 3 with no increase
in DNA yield observed at higher speeds.
Proteins were extracted at a concentrationof 1.08 mg/ml. Gel electrophoresis
indicated that the extracted genomic
DNA was of good quality with no shearing

Temperature vs Homogenization Time on the BM43634

Temperature (ºC)

Protein Extraction and Separation (cont.)
concentrations. 10 µl of each sample
was added to 10 ul of Laemmli sample
buffer (Bio-Rad) and heated at 95°C
for 5 minutes. The proteins were thenseparated by electrophoresis on a 4-20%
Tris-Glycine SDS polyacrylamide gel (BioRad) at 150 V for 45 minutes in Tris-Glycine
SDS running buffer (Bio-Rad). The gel wassilver stained using the ProteoSilver Stain
kit (Sigma-Aldrich) per the manufacturer’s
instructions and visualized on a GelDocEZ System.

32
Speed 130

Speed 228

Speed 3
Speed 426

Speed 524

Figure 1
Intratube temperature as afunction of homogenization
speed and processing time. Itwas observed that at speeds
below three the temperature
increase was neglible even after
processing for 75 seconds.

220102030

4050

607080Time (seconds)

Bead Mill 4 Speed Setting(Time = 60 sec)

Average DNA Concentration(ng/ul)

Average Protein
Concentration (mg/ml)
NA321.07

425.03NA522.63

1.08

Table 1 DNA and protein concentrations measured post homogenization.
observed as evident from the single
bands in lanes two through four
(Figure 2).

Figure 2 DNA agarose gel electrophoresisof E.coli K12 lysate. Lane 1: 100bp ladder.
Lane 2: Speed 3 DNA. Lane 3 DNA: Speed 4DNA. Lane 4: Speed 5 DNA.
The extraction procedure was further
evaluated by protein gel electrophoresis(Figure 3). Figure four indicates that
proteins with a broad molecular weight
range were extracted from the E. coli cells
following homogenization on the BeadMill 4.

Figure 3 Protein polyacrylamide gelelectrophoresis of E.coli K12 lysate. Lane 1:
Protein Ladder. Lane 2-4: E. coli K12 protein
extracts

Conclusion
The Bead Mill 4 is capable of disrupting E.
coli K12 cells up to a speed of 5 quickly
and with very little heat generated. TheDNA extraction was achieved at speeds inexcess of three with no detectable
shearing. Protein yields were significant
and the extraction procedure liberated
proteins with a broad molecular weight
range within forty seconds of processing.

Bead Mill 4: 15340164