eBook: Nucleic Acid Sample Extraction and Purification - 26

Resources
Application Note
PCR product purification with QIAquick 96
PCR Purification Kit and the VIAFLO 96
handheld electronic pipette
Introduction
QIAquick 96 PCR Purification Kit is suitable for purifying
up to 10 µg of material for downstream applications,
such as sequencing, cloning, labeling and microarrays.
The kit facilitates the removal of impurities like primers,
unincorporated nucleotides, buffers, salts, mineral oils,
agarose and enzymes. The vacuum-driven process is much
faster than centrifugation, and gives high, reproducible yields.
It is important to avoid cross-contamination in nucleic acid
Key benefits:
* VIAFLO 96 and VIAFLO 384 allow simultaneous pipetting of
up to 96 or 384 wells, respectively, maximize throughput of
PCR purification by allowing transfer samples and reagents
in a single step.
* The z-heights can be predefined, choosing the optimal value
to prevent accidental scratching of the well membrane for
more consistent results.
purification, and QIAGEN's column design is optimized
to limit carryover of contaminants. Although QIAquick 96
provides a high throughput solution, the elution, washing
and binding steps are very laborious and time consuming if
performed manually. With VIAFLO 96 handheld electronic
pipette, the hands-on time is reduced, as samples and
reagents can be transferred to all 96 wells at once. This
enables rapid and efficient, high throughput PCR clean-up.
* Custom programming of the PCR product clean-up
steps allows pipetting parameters, such as aspiration or
dispensing speeds, to be predefined. Prompt messages
guide the user through the entire pipetting protocol, which
is especially useful when several pre-wetting steps are
included.
* The VIAFLO 96 or VIAFLO 384's hands-free automatic
mode ensures that the PCR clean up protocols are
performed in the same way each time, maximizing
reproducibility.
Overview: how to purify PCR products with VIAFLO 96
Experimental set-up
This protocol describes how PCR products are purified
using a VIAFLO 96 handheld electronic pipette with a
two position stage and the QIAGEN QIAquick®
VIAFLO 96
96 PCR
Purification Kit. The following procedure is based on the kit
manufacturer's protocol for purification of 96 samples (up to
10 µg PCR products).
A 96 channel pipetting head (50-1250 µl) is used together
with 1250 µl SHORT, Low Retention, Sterile, Filter GripTips.
Customized VIALINK programs are provided to perform the
binding, washing and elution steps. Before starting, ethanol
(96-100 %) should be added to the Buffer PE concentrate.
PCR product purification
with QIAquick® 96 PCR
Purification Kit and the
VIAFLO 96 handheld
electronic pipette
VIAFLO_96_PCR_Product_Purification_V00
Application Note
Automation of the MACHEREY-NAGEL
NucleoSpin® 96 Tissue Kit on the ASSIST PLUS
pipetting robot
Introduction
MACHEREY-NAGEL's NucleoSpin®
96 nucleic acid
extraction and purification kits are known for producing
high quality nucleic acids. The NucleoSpin 96 Tissue kit
is designed for rapid purification of high molecular weight
genomic DNA from tissue samples, such as mouse and rat
tails, organ tissue, and animal or bacterial cells. The purified
DNA can be used directly for a variety of downstream
applications, including PCR, Southern blotting, genotyping,
NGS or any kind of enzymatic reaction. The NucleoSpin
Key benefits:
* 96 samples can be extracted in less than 1 hour by
processing 12 samples in parallel. Programs can be modified
if extraction of fewer samples is required.
* Automation of the liquid handling steps of the DNA purification
workflow with the ASSIST PLUS pipetting robot frees up time
for the user and increases reproducibility.
* Manual interventions are limited to operating the on/off button
of the vacuum, reagent reservoir exchange and placement
96 Tissue kit is convenient for high-throughput DNA
extractions, but the many pipetting steps required in the
manual processing of samples are tiring for users and
introduce the opportunity for pipetting errors at each step.
Performing the assay on the ASSIST PLUS pipetting robot
eliminates the possibility of pipetting errors and provides
straightforward semi-automated sample extraction,
eliminating the possibility of pipetting errors and reducing
the strain of repetitive pipetting.
of the elution plate within the vacuum manifold. Instructions
for these steps are provided on the pipette's bright screen,
ensuring the user does not lose track of the process.
* The benefit of the NucleoSpin 96 Tissue kit over other
options is the inclusion of a specially designed wash plate,
inserted into the manifold beneath the binding plate. The
wash plate prevents cross-contamination of samples during
the vacuum steps.
Overview: How to automate the NucleoSpin 96 Tissue kit
This protocol describes the automation of the pipetting steps
of the MACHEREY-NAGEL NucleoSpin 96 Tissue kit are
automated on the ASSIST PLUS pipetting robot. All liquids
are transferred automatically, with manual interventions only
required for turning the vacuum on and off, loading reagent
reservoir 1 and 2, and preparing the elution plate.
The ASSIST PLUS operates a 12 channel VIAFLO 1250 μl
electronic pipette with 1250 μl Sterile, Filter GripTips.
Before starting, the indicated volume of Proteinase Buffer
PB should be added to the lyophilized Proteinase K (see
MACHEREY-NAGEL protocol) and ethanol (96-100 %)
should be added to the Wash Buffer B5 Concentrate.
ASSIST PLUS
ASSIST PLUS_MN_NucleoSpin_96_Tissue_Application_V00
1/8
Automation of the
MACHEREY-NAGEL
NucleoSpin® 96 Tissue
Kit on the ASSIST PLUS
pipetting robot
Application Note
Transfection-grade plasmid DNA purification
using MACHEREY-NAGEL's NucleoSpin® 96
Plasmid Transfection-grade kit and
NucleoVac 96 Vacuum Manifold
Introduction
The use of silica membrane-based DNA purification kits is
a convenient way to prepare high quality, transfection-grade
plasmid DNA samples for cloning, sequencing and restriction
analysis or for more sensitive applications, such as transfection
of standard cell lines. In the latter case, transfection efficiency
is negatively impacted by endotoxins, and so it is important that
levels are low. MACHEREY-NAGEL has developed a novel
technology to reduce endotoxin content. Its NucleoSpin®
96
Plasmid Transfection-grade kit is designed for high throughput
purification of high-copy plasmid DNA from E. coli in a 96 well
plate format. The miniprep protocol is based on alkaline lysis,
and is optimized for the purification of plasmid DNA from
1-5 ml of bacterial culture. However, it is a time-consuming
step in genetic analyses.
Key benefits:
* Use of MACHEREY-NAGEL's NucleoSpin 96 Plasmid
Transfection-grade kit and NucleoVac 96 Vacuum Manifold
is a proven approach for high throughput purification of
plasmid DNA.
* Automation of the pipetting steps of the miniprep workflow
with the ASSIST PLUS pipetting robot offers more handsfree
time for the user and increases reproducibility.
Step-by-step procedure:
The ASSIST PLUS pipetting robot is used to automate the
pipetting steps of the MACHEREY-NAGEL NucleoSpin 96
Plasmid Transfection-grade kit purification protocol. The
liquid handling platform guides the user whenever manual
interventions are required during the process. The following
procedure is based on the kit manufacturer's protocol for
purification of 96 samples.
Bacteria are first cultivated at 37 °C following MACHEREYNAGEL's
recommendations, either in a square-well block
or tubes. For the present protocol, the bacterial cultures are
grown in tubes and transferred into a square-well block. The
cultures are centrifuged for 10 min at 1000 x g to pellet the
bacteria. The supernatant is discarded, and the residual
medium removed by tapping the plate upside down on a
clean paper sheet or soft tissue.
ASSIST PLUS_MN_NucleoSpin_Plasmid_TF_V00
ASSIST PLUS
The ASSIST PLUS pipetting robot, together with a VIAFLO
12 channel 1250 µl electronic pipette, allows automation of
the MACHEREY-NAGEL plasmid DNA isolation protocol.
The system performs all the pipetting steps, guaranteeing
perfect and reproducible liquid handling while protecting the
user from repetitive strain injuries. The pipette guides the
user through each manual intervention in the purification
process, ensuring an error-free workflow. Together,
MACHEREY-NAGEL's NucleoSpin 96 Plasmid Transfectiongrade
kit and INTEGRA's ASSIST PLUS pipetting robot
provide researchers with an easy and efficient way to extract
and purify plasmid DNA of up to 96 samples with minimal
hands-on time.
* 96 samples can be purified in less than 45 minutes, by
processing 12 samples in parallel.
* In combination with the ASSIST PLUS, the VIAFLO
electronic pipettes provide unmatched ergonomics.
* The ASSIST PLUS performs all the pipetting steps of
the protocol, and guides the user through each manual
intervention.
Transfection-grade
plasmid DNA purification
using MACHEREY-NAGEL's
NucleoSpin® 96 Plasmid
Transfection-grade
kit and NucleoVac 96
Vacuum Manifold
Application Note
Plasmid DNA purification using
MACHEREY-NAGEL's NucleoSpin® 96 Plasmid
kit and NucleoVac 96 Vacuum Manifold
Introduction
The use of silica membrane-based DNA purification kits
is a convenient way to prepare plasmid DNA samples for
cloning or subsequent sequencing or restriction analysis.
MACHEREY-NAGEL's NucleoSpin 96 Plasmid kit is designed
for high throughput purification of high-copy plasmid DNA
from E. coli in a 96 well plate format. The miniprep protocol
is based on alkaline lysis and typically yields 5-15 µg of
plasmid DNA from 1.5 ml overnight cultures. It is, however, a
time-consuming step in genetic analysis. The ASSIST PLUS
pipetting robot, together with a VIAFLO 12 channel 1250 µl
Key benefits:
* MACHEREY-NAGEL's NucleoSpin 96 Plasmid kit and
NucleoVac 96 Vacuum Manifold are a proven technique for
high throughput plasmid DNA purification.
* Automation of the pipetting steps of the miniprep workflow
with the ASSIST PLUS pipetting robot allows for more
hands-free time for the user and increased reproducibility.
* 96 samples can be purified in less than 45 minutes by
Step-by-step procedure:
Experimental set-up
The ASSIST PLUS pipetting robot is used to automate the
pipetting steps of the MACHEREY-NAGEL NucleoSpin 96
Plasmid kit purification protocol. The liquid handling platform
guides the user whenever manual interventions are required
during the process. The following procedure is based on the
kit manufacturer's protocol for purification of 96 samples.
Bacteria are first cultivated at 37 °C following MACHEREYNAGEL's
recommendations, either in a Square-well Block
or tubes. For the present protocol, the bacterial cultures are
grown in tubes and transferred into a Square-well Block.
Centrifuge the bacterial cultures for 10 min at 1000 x g to
pellet the bacteria. Discard the supernatant and remove the
residual medium by tapping the plate upside down on a clean
paper sheet or soft tissue.
Plasmid DNA purification_NucleoSpin_V00
electronic pipette, allows automation of the MACHEREYNAGEL
plasmid DNA isolation protocol. The system performs
all the pipetting steps, guaranteeing perfect and reproducible
liquid handling while protecting the user from repetitive strain
injuries. The pipette guides the user through each manual
intervention in the purification process, ensuring an error-free
workflow. Together, MACHEREY-NAGEL's NucleoSpin 96
Plasmid kit and INTEGRA's ASSIST PLUS pipetting robot
provide researchers with an easy and efficient way to extract
and purify plasmid DNA with minimal hands-on time.
processing 12 samples in parallel. In combination with the
ASSIST PLUS, the VIAFLO electronic pipettes provide
unmatched ergonomics, eliminating the need to hold the
pipette during the pipetting steps.
* The ASSIST PLUS performs all the pipetting steps of
the protocol and guides the user through each manual
intervention.
Plasmid DNA purification
of MACHEREY-NAGEL's
NucleoSpin® 96 Plasmid
kit and NucleoVac 96
Vacuum Manifold
ASSIST PLUS
The ASSIST PLUS pipetting robot operates a VIAFLO 12
channel 1250 µl electronic pipette with 1250 µl Sterile,
Filter GripTips.
1/9
ASSIST_PLUS_HPV_V00
1/5
Application Note
Automation of the MACHEREY-NAGEL
NucleoSpin® 96 RNA Kit on the ASSIST PLUS
pipetting robot
Introduction
MACHEREY-NAGEL NucleoSpin®
96 nucleic acid extraction
and purification kits are known to yield high quality and
abundant nucleic acids. The 96 well silica membrane plate
format used in the NucleoSpin 96 RNA kit is convenient
for extracting RNA from cell cultures or tissues in high
throughput laboratories, but the many pipetting steps
required for manual processing of samples are tiring for
Key benefits:
* 96 samples can be extracted per run. If extraction of fewer
samples is desired, programs may be modified. The
ASSIST PLUS pipetting robot is equipped with a 12 channel
VIAFLO electronic pipette, making the process even faster.
* An external plate shaker and MACHEREY-NAGEL Vacuum
Manifold are placed directly on the deck of the ASSIST
PLUS. This eliminates the need to transport plates from the
liquid handler to these instruments and back.
* Manual interventions are limited to operating the on and
off button of the shaker and vacuum, changing the reagent
reservoir, and placing the elution plate in the vacuum
users, and introduce the possibility of pipetting errors at
each step. Performing the assay on the ASSIST PLUS
pipetting robot eliminates this possibility, and reduces the
strain of repetitive pipetting by providing semi-automated
sample extraction. The processing of 96 samples with the
ASSIST PLUS pipetting robot is possible in less than 1 hour.
manifold. Instructions for these interventions are provided
on the pipette's bright screen, ensuring that the user does
not lose track of the process.
* Benefits of the MACHEREY-NAGEL NucleoSpin 96 RNA
kit over other kits include the addition of a DNA digestion
step to ensure that the eluted RNA is free from DNA
contamination, and the inclusion of a specially designed
wash plate inserted into the manifold beneath the binding
plate. This wash plate prevents cross contamination of
samples during the vacuuming steps.
Overview: How to automate the MACHEREY-NAGEL NucleoSpin 96 RNA Kit
ASSIST PLUS
cells or 30 mg tissue). All liquids are transferred
In this protocol, the pipetting steps of the MACHEREYNAGEL
NucleoSpin 96 RNA kit are automated on the
ASSIST PLUS pipetting robot. With the NucleoSpin 96
RNA kit RNA can be isolated from cells or tissues (up to
2*106
automatically, with manual interventions only needed for
turning the shaker and vacuum on and off, loading reagent
reservoir 2, and preparing the elution plate.
The ASSIST PLUS operates a 12 channel VIAFLO 1250 μl
electronic pipette with 1250 μl Sterile, Filter GripTips.
Before beginning, RNase-free rDNase is reconstituted (see
MACHEREY-NAGEL protocol), rDNase reaction mixture
is prepared, and ethanol (96-100 %) is added to the wash
buffers RA3 and RA4.
ASSIST PLUS_MN_NucleoSpin_96_RNA_V00
Automation of the
MACHEREY-NAGEL
NucleoSpin® 96 RNA
Extraction Kit on
the ASSIST PLUS
pipetting robot
Application Note
Rapid detection of HPV high-risk types
16 and 18 in cervical cancer screening
Introduction
Human papillomavirus (HPV) infections are known to be
a high-risk factor in developing various types of diseases.
Crucially, almost all cervical cancer cases are related to
oncogenic HPV infections. The disease remains the fourth
most common cancer in women, with an estimated 530,000
new cases per year, and approximately 275,000 deaths
worldwide.
There are several types of high-risk oncogenic human
papillomaviruses, including HPV types 16 and 18 that are
responsible for around 70 % of cervical cancers worldwide.
However, HPV-related cervical cancer could be prevented
with regular screening. HPV testing is more sensitive than the
Pap smear. As a result, many countries are now considering
the use of HPV testing either to complement smear testing
or as a primary test. Consequently, the demand for HPV
testing is increasing, driving a need for automation while still
keeping the process simple.
The High-risk Human Papillomavirus DNA Fluorescence
Diagnostic Kit (Sansure Biotech Inc.) is a nucleic acid
extraction and amplification test for the detection of the highrisk
oncogenic HPV types 16 and 18. It does not require
time-consuming DNA purification steps prior to the PCR setup,
allowing for faster results compared to most PCR tests
as well as increased sample processing capacity.
Rapid detection of
HPV high-risk types
16 and 18 in cervical
cancer screening
Key benefits:
* Automating VOYAGER adjustable tip spacing pipettes on
the ASSIST PLUS pipetting robot provides a fast, efficient
and error-free, sample preparation method for 96 samples.
* The system increases sample throughput, eliminates
the risk of reformatting errors and minimizes manual
intervention.
* The method described in this application note is
ready for molecular diagnostic labs to use, making the
Step-by-step procedure:
Experimental set-up
The ASSIST PLUS pipetting robot is used to automate
the pipetting steps of the Sansure High-risk Human
Papillomavirus DNA Fluorescence Diagnostic Kit.
1 ml of each sample is collected and placed in 1.5 ml
microcentrifuge tubes before centrifugation for 5 minutes at
12,000 x g.
The ASSIST PLUS pipetting robot operates a VOYAGER
8 channel 50 µl adjustable tip spacing pipette with 125 µl
Sterile, Filter GripTips.
implementation of high-risk oncogenic HPV testing fast
and straightforward, with no additional set-up steps or
programming required by the user.
* The Sansure High-risk Human Papillomavirus DNA
Fluorescence Diagnostic Kit is an established product
that enables DNA testing with a 15-minute lysis,
minimizing the time until results are provided to patients.
ASSIST PLUS
26
https://www.integra-biosciences.com/en/applications/pcr-product-purification-qiaquickr-96-pcr-purification-kit-and-viaflo-96-handheld-electronic-pipette https://www.integra-biosciences.com/en/applications/automation-macherey-nagel-nucleospinr96-tissue-kit-assist-plus-pipetting-robot https://www.integra-biosciences.com/united-kingdom/en/applications/transfection-grade-plasmid-dna-purification-using-macherey-nagels-nucleospinr-96-plasmid-transfection-grade-kit-and-nucleovac-96-vacuum-manifold https://www.integra-biosciences.com/united-kingdom/en/applications/automation-macherey-nagel-nucleospinr-96-rna-extraction-kit-assist-plus-pipetting-robot https://www.integra-biosciences.com/en/applications/plasmid-dna-purification-macherey-nagels-nucleospinr-96-plasmid-kit-and-nucleovac-96-vacuum-manifold https://www.integra-biosciences.com/en/applications/rapid-detection-hpv-high-risk-types-16-and-18-cervical-cancer-screening
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