ChargeSwitch® gDNA Tissue Kits

ChargeSwitch® gDNA Tissue Kits
ChargeSwitch® gDNA Tissue Kits
For purification of genomic DNA from tissue
samples
Catalog nos. CS11203 and CS11204
Rev. date: 20 May 2010
Manual part no. 25-0817
MAN0000505
Corporate Headquarters
5791 Van Allen Way
Carlsbad, CA 92008
T: 1 760 603 7200
F: 1 760 602 6500
E: [email protected]
For country-specific contact information visit our web site at www.invitrogen.com
User Manual
ii
Table of Contents
Kit Contents and Storage .................................................................................... iv
Introduction ........................................................................................ 1
Product Overview ..................................................................................................1
Experimental Outline ............................................................................................3
Methods............................................................................................... 4
General Information ..............................................................................................4
Isolating Genomic DNA Using the Micro Tissue Kit.......................................7
Isolating Genomic DNA Using the Mini Tissue Kit.......................................13
Troubleshooting ...................................................................................................18
Appendix ........................................................................................... 21
Accessory Products..............................................................................................21
Technical Support ................................................................................................22
Purchaser Notification.........................................................................................24
iii
Kit Contents and Storage
Types of Kits
This manual is supplied with the following products.
Product
Catalog no.
®
Shipping and
Storage
ChargeSwitch gDNA Micro Tissue Kit
CS11203
ChargeSwitch® gDNA Mini Tissue Kit
CS11204
All components of the ChargeSwitch® gDNA Tissue Kits
are shipped at room temperature. Upon receipt, store the
Proteinase K and RNase A at 4C. Store all other
components at room temperature.
All components are guaranteed stable for 6 months if
stored properly.
Kit Contents
The components supplied in the ChargeSwitch® gDNA
Tissue Kits are listed below. The reagents supplied are
sufficient to perform 25 (Mini Tissue) or 50 (Micro Tissue)
purifications.
Note: Some reagents in the kit may be provided in excess of the
amount needed.
Component
Micro Tissue
55 mL
--
ChargeSwitch Lysis Buffer (L15)
--
50 mL
ChargeSwitch ® Magnetic Beads
3 × 1 mL
2 × 1 mL
Proteinase K (20 mg/mL in 50 mM
Tris-HCl, pH 8.5, 5 mM CaCl2, 50%
glycerol)
500 μL
500 μL
RNase A (5 mg/mL in 10 mM TrisHCl, pH 8.5, 10 mM EDTA)
250 μL
250 μL
ChargeSwitch® Purification Buffer (N5) 10 mL
10 mL
®
ChargeSwitch Lysis Buffer (L13)
®
®
ChargeSwitch Wash Buffer (W12)
50 mL
100 mL
ChargeSwitch® Elution Buffer (E5;
10 mM Tris-HCl, pH 8.5)
10 mL
10 mL
Intended Use
iv
Amount
Mini Tissue
For research use only. Not intended for any animal or
human therapeutic or diagnostic use.
Introduction
Product Overview
Description of
the System
The ChargeSwitch® gDNA Mini and Micro Tissue Kits allow
rapid and efficient purification of genomic DNA from mini
(10–25 mg) or micro (3–5 mg) quantities of tissue,
respectively. After preparing the lysates, you may purify
DNA in less than 15 minutes using the ChargeSwitch®
Technology. For more information about the ChargeSwitch®
Technology, see page 2.
Intended Use
for the Kits
The ChargeSwitch® gDNA Tissue Kits are designed to allow
isolation of genomic DNA from the following sources. The
purified genomic DNA is suitable for use in downstream
applications including PCR, restriction enzyme digestion,
and Southern blotting.
Advantages

Micro-Tissue Kit: Purifies up to 5 μg of genomic DNA
from 1–2 mm diameter mouse ear clips or 3–5 mg of
tissue. This sample size is suitable for genomic DNA
purification from micro-dissected or laser capture microdissected samples.

Mini-Tissue Kit: Purifies up to 30 μg of genomic DNA
from 0.5 cm mouse tail tips or approximately 25 mg of
tissue. For spleen tissue, reduce tissue size to 10 mg.
Use of the ChargeSwitch® gDNA Tissue Kits to isolate
genomic DNA provides the following advantages:

Uses a magnetic bead-based technology to isolate
genomic DNA without the need for hazardous
chemicals, centrifugation, or vacuum manifolds

Rapid and efficient purification of genomic DNA from
micro or mini quantities of tissue in less than 15 minutes
following sample preparation and lysis

Simple lysis of tissues with Proteinase K without the
need for any mechanical lysis

Minimal contamination with RNA

The purified genomic DNA demonstrates improved
downstream performance in applications including PCR,
restriction enzyme digestion, and Southern blotting.
Continued on next page
1
Product Overview, Continued
System
Specifications
Mini-Tissue
Micro-Tissue
Starting Material:
10–25 mg
3–5 mg
Elution Volume:
250 μL
150 μL
DNA Yield:
Up to 30 μg
Up to 5 μg
DNA Size:
> 20 kb
> 20 kb
The ChargeSwitch® Technology (CST®) is a novel magnetic
The
®
ChargeSwitch bead-based technology that provides a switchable surface
charge dependent on the pH of the surrounding buffer to
Technology
facilitate nucleic acid purification. In low pH conditions, the
CST® beads have a positive charge that binds the negatively
charged nucleic acid backbone (see figure below). Proteins
and other contaminants are not bound and are simply
washed away in an aqueous wash buffer. To elute nucleic
acids, the charge on the surface of the bead is neutralized by
raising the pH to 8.5 using a low salt elution buffer (see
figure below). Purified DNA elutes instantly into this elution
buffer, and is ready for use in downstream applications.
Low pH
ChargeSwitch® Bead Binding Capacity:
Magnetic Bead Bead Size:
Specifications
2
High pH
5–10 μg genomic DNA per mg
< 1 μm
Bead Concentration:
25 mg/mL
Storage Buffer:
10 mM MES, pH 5.0, 10 mM
NaCl, 0.1% Tween 20
Experimental Outline
Introduction
The figure below illustrates the basic steps necessary to
purify genomic DNA from tissues using the ChargeSwitch®
gDNA Tissue Kits.
Lyse sample
Bead charge
Bind DNA to ChargeSwitch®
magnetic beads
Wash beads containing DNA
to remove contaminants
Elute DNA from beads
Charge on
pH < 6.0
Charge on
pH = 7.0
Charge off
pH = 8.5
3
Methods
General Information
User Supplied
Materials
MagnaRack™

A magnetic separation rack suitable for use with 1.5 mL
microcentrifuge tubes (see below)

Sterile, 1.5 mL microcentrifuge tubes

Vortex mixer

20 μL, 200 μL, and 1 mL sterile, pipette tips

Water bath at 55C
The MagnaRack™ available from Invitrogen (see page 21) is a
two-piece magnetic separation rack for use in protocols with
magnetic beads. The MagnaRack™ consists of a magnetic
base station and a removable tube rack. The tube rack can
hold up to 24 microcentrifuge tubes. The tube rack fits onto
the magnetic base station in two different positions,
associating the row of 12 neodymium magnets with a single
row of 12 tubes for simple ‘on the magnet’ and ‘off the
magnet’ sample processing (see figure below). For more
information, see www.invitrogen.com or call Technical
Support (page 22).
Continued on next page
4
General Information, Continued
Safety
Information
Follow the safety guidelines below when using the
ChargeSwitch® gDNA Tissue Kit.

Treat all reagents supplied in the kit as potential
irritants.

Always wear a suitable lab coat, disposable gloves, and
protective goggles.

If a spill of the buffers occurs, clean with a suitable
laboratory detergent and water. If the liquid spill
contains potentially infectious agents, clean the affected
area first with laboratory detergent and water, then
with 1% (v/v) sodium hypochlorite or a suitable
laboratory disinfectant.
Follow the guidelines below when handling the
Handling the
ChargeSwitch® ChargeSwitch® magnetic beads.
Magnetic

Do not freeze the beads as this irreparably damages
Beads
them. Store the beads at room temperature.

Always keep the beads in solution. Do not allow them
to dry out as this renders them non-functional.

When using the beads, resuspend thoroughly in the
storage buffer by vortexing before removal.

Discard beads after use. Do not reuse.
Continued on next page
5
General Information, Continued
Elution Buffer
ChargeSwitch® Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5)
is supplied with the kit for eluting the DNA from the
ChargeSwitch® Magnetic Beads. For best results, use Elution
Buffer (E5) to elute the DNA. Alternatively, TE Buffer, pH
8.5–9.0 is acceptable. Note that the pH must be 8.5–9.0,
otherwise the DNA will not elute. Do not use water for
elution.
The protocol recommends eluting the genomic DNA in
150 μL (Micro Kit) or 250 μL (Mini Kit) of ChargeSwitch®
Elution Buffer (E5). You may vary the amount of
ChargeSwitch® Elution Buffer (E5) used to obtain genomic
DNA in the desired final concentration. For best results,
always use a volume of ChargeSwitch® Elution Buffer (E5)
that is equal to or greater than the volume of
ChargeSwitch® Magnetic Beads used in the protocol. If the
volume of ChargeSwitch® Elution Buffer (E5) is lower than
the volume of beads used, DNA elution is incomplete. You
may need to perform a second elution to recover all DNA.
6
Isolating Genomic DNA Using the Micro
Tissue Kit
Introduction
Starting
Material
This section provides guidelines and instructions to isolate
genomic DNA from micro quantities of tissue using the
ChargeSwitch® gDNA Micro Tissue Kit (Catalog no.
CS11203).
Use this procedure to isolate genomic DNA from:

1–2 mm mouse ear clips

3–5 mg tissue
If you wish to isolate genomic DNA from larger amounts of
tissue or from mouse tail tips, see Isolating Genomic DNA
Using the Mini Tissue Kit, page 13.
Materials
Needed

Tissue sample(s) (see above)

MagnaRack™ (see page 21) or other magnetic separation
rack

Sterile 1.5 mL microcentrifuge tubes

Vortex mixer

Sterile pipette tips (20 μL, 200 μL, and 1 mL)

Water bath
Components Supplied with the Kit

ChargeSwitch® Lysis Buffer (L15)

Proteinase K

RNase A

ChargeSwitch® Magnetic Beads

ChargeSwitch® Purification Buffer (N5)

ChargeSwitch® Wash Buffer (W12)

ChargeSwitch® Elution Buffer (E5) or TE Buffer (not
supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)
Continued on next page
7
Isolating Genomic DNA Using the Micro
Tissue Kit, Continued
Before
Starting
Perform the following before beginning:

Set a water bath at 55C.

Prepare Lysis Mix: For each sample, mix 1 mL of
ChargeSwitch® Lysis Buffer (L15) and 10 μL of Proteinase
K to prepare the Lysis Mix. When isolating DNA from
multiple samples, scale up the volume of reagents used
and prepare a master Lysis Mix.
Note: The ChargeSwitch® Lysis Buffer may appear slightly
cloudy. If cloudy, shake the bottle before use until the solution
becomes clear.
Preparing the
Tissue Lysate
Follow the procedure below to prepare a lysate from the
tissue sample.
1.
Place the tissue sample into a sterile microcentrifuge
tube.
2.
Add 1 mL of Lysis Mix (see above) to the tube. Ensure
that the tissue is completely immersed in the Lysis
Buffer.
3.
Vortex for 10–15 seconds to mix.
4.
Incubate the sample overnight at 55C until lysis is
complete.
Note: The length of the incubation step can be shortened to
1-2 hours by vortexing the sample at least 4 times during this
period.
5.
Add 5 μL of RNase A to the lysate. Pipet up and down
gently 5 times or until a homogeneous solution is
obtained.
Important: Use a 1 mL pipette tip set to 900 μL to mix the
sample. Make sure that the tip is submerged, and pipet up and
down gently to avoid forming bubbles as this may result in
shearing of the DNA.
6.
Incubate at room temperature for 5 minutes.
7.
Proceed to Binding DNA, next page.
Continued on next page
8
Isolating Genomic DNA Using the Micro
Tissue Kit, Continued
Binding DNA
Follow the procedure below to bind the DNA to the
ChargeSwitch® Magnetic Beads.
1.
Vortex the tube containing the ChargeSwitch® Magnetic
Beads to fully resuspend and evenly distribute the beads
in the storage buffer. Make sure that all of the solution
containing the beads is at the bottom of the tube.
2.
Add 200 μL of ChargeSwitch® Purification Buffer (N5) to
the digested tissue sample (from Step 6, previous page)
and pipet up and down gently twice to mix.
Note: Adding the ChargeSwitch® Purification Buffer lowers the
pH of the sample, and optimizes the binding conditions.
3.
Add 40 μL of ChargeSwitch® Magnetic Beads (from
Step 1) to the sample and pipet up and down gently
5 times to mix.
4.
Incubate at room temperature for 1 minute to allow the
DNA to bind to the ChargeSwitch® Magnetic Beads.
5.
Place the sample in the MagnaRack™ for 1 minute or until
the beads have formed a tight pellet.
6.
Without removing the tube from the MagnaRack™,
carefully remove the supernatant and discard. Take care
not to disturb the pellet of beads by angling the pipette
such that the tip is pointed away from the pellet (see
figure below).
Pipette tip
Magnetic pellet
7.
Proceed immediately to Washing DNA, next page.
Continued on next page
9
Isolating Genomic DNA Using the Micro
Tissue Kit, Continued
Washing DNA
1.
Remove the tube containing the pelleted magnetic beads
from the MagnaRack™ (Step 6, previous page). There
should be no supernatant in the tube.
2.
Add 1 mL of ChargeSwitch® Wash Buffer (W12) to the
tube and pipet up and down gently twice to resuspend
the magnetic beads.
Important: Use a 1 mL pipette tip set to 900 μL to mix the
sample. Make sure that the tip is submerged, and pipet up and
down gently to avoid forming bubbles.
3.
Place the sample in the MagnaRack™ for 1 minute or until
the beads have formed a tight pellet.
4.
Without removing the tube from the MagnaRack™,
carefully remove the supernatant and discard. Take care
not to disturb the pellet of beads by angling the pipette
such that the tip is pointed away from the pellet (see
figure on page 9).
5.
Repeat Steps 1–4.
6.
Proceed to Eluting DNA, next page.
Continued on next page
10
Isolating Genomic DNA Using the Micro
Tissue Kit, Continued
Eluting DNA
1.
Remove the tube containing the pelleted magnetic beads
from the MagnaRack™ (Step 5, previous page). There
should be no supernatant in the tube.
2.
Add 150 μL of ChargeSwitch® Elution Buffer (E5) (or TE
Buffer, pH 8.5) to the tube and pipet up and down gently
10 times to resuspend the magnetic beads.
Important: Do not use water for elution. The DNA will not elute
due to the poor buffering capacity of water.
3.
Incubate at room temperature for 5 minutes.
Tip: For maximum yield, mix the suspension of beads (by
pipetting up and down gently) half way through the incubation.
Incubating the sample at 55C may also improve yield.
4.
Place the sample in the MagnaRack™ for 1 minute or until
the beads have formed a tight pellet.
5.
Without removing the tube from the MagnaRack™,
carefully remove the supernatant containing the DNA to
a sterile microcentrifuge tube. Take care not to disturb
the pellet of beads by angling the pipette such that the tip
is pointed away from the pellet (see figure on page 9).
Note: If the eluate containing the DNA is discolored, repeat
Steps 5–6.
Storing DNA
6.
Discard the used magnetic beads. Do not reuse the beads.

Store the purified DNA at –20C or use immediately for
the desired downstream application.

Avoid repeatedly freezing and thawing DNA. Store the
purified DNA at 4C for short-term use or aliquot the
DNA and store at –20C for long-term storage.
Continued on next page
11
Isolating Genomic DNA Using the Micro
Tissue Kit, Continued
Quantitating
DNA Yield
You may estimate the yield of purified genomic DNA by
checking the UV absorbance at 260 nm or using one of the
Quant-iT™ DNA Assay Kits.
UV Absorbance
1.
Measure the A260 of the solution using a spectrophotometer blanked against 10 mM Tris-HCl, pH 8.5.
2.
Calculate the amount of DNA using the formula:
DNA (μg) = A260  50 μg/(A260 × 1 mL) × dilution factor
× total sample volume (mL)
For DNA, A260 = 1 for a 50 μg/mL solution measured in a
cuvette with an optical path length of 1 cm.
Quant-iT™ DNA Assay Kits
The Quant-iT™ DNA Assay Kits (see page 21 for ordering
information) provide a rapid, sensitive, and accurate
method for dsDNA quantitation with minimal interference
from RNA, protein, ssDNA (primers), or other common
contaminants that affect UV absorbance. Each kit contains a
state-of-the-art quantitation reagent, pre-diluted standards
for a standard curve, and a pre-made buffer to allow
fluorescence-based DNA quantitation. For more
information, see www.invitrogen.com or call Technical
Support (page 22).
12
Isolating Genomic DNA Using the Mini
Tissue Kit
Introduction
This section provides guidelines and instructions to isolate
genomic DNA from mini quantities of tissue using the
ChargeSwitch® gDNA Mini Tissue Kit (Catalog no. CS11204).
Starting
Material
Use this procedure to isolate genomic DNA from:

0.5 cm mouse tail tips

Up to 25 mg tissue (up to 10 mg only for spleen tissue)
If you wish to isolate genomic DNA from smaller amounts
of tissue or from mouse ear clips, see Isolating Genomic
DNA Using the Micro Tissue Kit, page 7.
Materials
Needed

Tissue sample(s) (see above)

1 mL mini glass homogenizer (if isolating DNA from
tissues other than mouse tail tips; Fisher, Cat no.
NC9051099)

MagnaRack™ (see page 21) or other magnetic separation
rack

Sterile 1.5 mL microcentrifuge tubes

Vortex mixer

Sterile pipette tips (20 μL, 200 μL, and 1 mL)

Water bath
Components Supplied with the Kit

ChargeSwitch® Lysis Buffer (L13)

Proteinase K

RNase A

ChargeSwitch® Magnetic Beads

ChargeSwitch® Purification Buffer (N5)

ChargeSwitch® Wash Buffer (W12)

ChargeSwitch® Elution Buffer (E5) or TE Buffer (not
supplied; 10 mM Tris-HCl, 1 mM EDTA, pH 8.5)
Continued on next page
13
Isolating Genomic DNA Using the Mini
Tissue Kit, Continued
Preparing the
Tissue Lysate
Follow the procedure below to prepare a cell lysate from the
tissue sample.
1.
Set a water bath at 55C.
2.
Prepare the tissue samples as follows:

For mouse tail tips: Place the tissue sample into a
sterile 1.5 mL microcentrifuge tube. Add 1 mL of
ChargeSwitch® Lysis Buffer (L13) to the tube. Ensure
that the tissue is completely immersed in the Lysis
Buffer.

For other tissues: Place the tissue in a 1 mL mini
glass homogenizer. Add 1 mL of ChargeSwitch®
Lysis Buffer (L13) to the tube. Homogenize with
approximately 8 strokes until the tissue is welldispersed. Transfer the homogenized tissue to a
sterile 1.5 mL microcentrifuge tube.
3.
Add 20 μL of Proteinase K to the tube.
4.
Vortex or invert the tube for 10–15 seconds to mix.
5.
Incubate the sample for 1.5–3 hours at 55C until lysis is
complete. The lysate should appear clear.
Note: Some tissues (e.g. brain) can produce a cloudy lysate at
55C. This does not adversely affect purification.
6.
For samples containing bone or hair: Centrifuge the
sample for 1 minute at 13,000 rpm. Transfer the
supernatant to a clean tube.
For all other samples: Proceed directly to Step 7.
7.
Add 10 μL of RNase A to the lysate. Invert the tube to
mix the solution.
8.
Incubate at room temperature for 2 minutes.
9.
Proceed to Binding DNA, next page.
Continued on next page
14
Isolating Genomic DNA Using the Mini
Tissue Kit, Continued
Binding DNA
Follow the procedure below to bind the DNA to the
ChargeSwitch® Magnetic Beads.
1.
Vortex the tube containing the ChargeSwitch® Magnetic
Beads to fully resuspend and evenly distribute the beads
in the storage buffer. Make sure that all of the solution
containing beads is at the bottom of the tube.
2.
Add 120 μL of ChargeSwitch® Magnetic Beads (from
Step 1) to the digested tissue sample (from Step 8,
page 14) and pipet up and down gently 5 times to mix.
Important: Use a 1 mL pipette tip set to 900 μL to mix the
sample. Make sure that the tip is submerged, and pipet up and
down gently to avoid forming bubbles as this may result in
shearing of the DNA.
3.
Add 100 μL of ChargeSwitch® Purification Buffer (N5) to
the sample and pipet up and down gently (with a 1 mL
pipette tip) 10 times to mix.
Note: Adding the ChargeSwitch® Purification Buffer lowers the
pH of the sample, and allows DNA to bind to the beads.
4.
Place the sample in the MagnaRack™ for 2 minutes or
until the beads have formed a tight pellet.
Note: Some tissues (e.g. spleen) may produce a viscous
supernatant that retards the movement of the beads, prolonging
separation times. If this occurs, leave the sample in the
MagnaRack™ until the beads have formed a tight pellet and the
supernatant is clear.
5.
Without removing the tube from the MagnaRack™,
carefully remove the supernatant and discard. Take care
not to disturb the pellet of beads by angling the pipette
such that the tip is pointed away from the pellet (see
figure below).
Pipette tip
Magnetic pellet
6.
Proceed immediately to Washing DNA, next page.
Continued on next page
15
Isolating Genomic DNA Using the Mini
Tissue Kit, Continued
Washing DNA
1.
Remove the tube containing the pelleted magnetic beads
from the MagnaRack™ (Step 5, previous page). There
should be no supernatant in the tube.
2.
Add 1 mL of ChargeSwitch® Wash Buffer (W12) to the
tube and pipet up and down gently twice to resuspend
the magnetic beads.
Important: Use a 1 mL pipette tip set to 900 μL to mix the
sample. Make sure that the tip is submerged, and pipet up and
down gently to avoid forming bubbles.
3.
Place the sample in the MagnaRack™ for 1 minute or until
the beads have formed a tight pellet.
4.
Without removing the tube from the MagnaRack™,
carefully remove the supernatant and discard. Take care
not to disturb the pellet of beads by angling the pipette
such that the tip is pointed away from the pellet (see
figure on page 15).
5.
Repeat Steps 1–4.
6.
Proceed to Eluting DNA, next page.
Continued on next page
16
Isolating Genomic DNA Using the Mini
Tissue Kit, Continued
Eluting DNA
1.
Remove the tube containing the pelleted magnetic beads
from the MagnaRack™ (Step 5, previous page). There
should be no supernatant in the tube.
2.
Add 250 μL of ChargeSwitch® Elution Buffer (E5) (or TE
Buffer, pH 8.5) to the tube and pipet up and down gently
twice to resuspend the magnetic beads.
Important: Do not use water for elution. The DNA will not elute
due to the poor buffering capacity of water.
3.
Incubate at 55C for 5 minutes.
4.
Place the sample in the MagnaRack™ for 2 minutes or
until the beads have formed a tight pellet.
5.
Without removing the tube from the MagnaRack™,
carefully remove the supernatant containing the DNA to
a sterile microcentrifuge tube. Take care not to disturb
the pellet of beads by angling the pipette such that the tip
is pointed away from the pellet (see figure on page 15).
Note: If the eluate containing the DNA is discolored, repeat
Steps 4–5.
Storing DNA
Quantitating
DNA Yield
6.
Discard the used magnetic beads. Do not reuse the beads.

Store the purified DNA at –20C or use immediately for
the desired downstream application.

Avoid repeatedly freezing and thawing DNA. Store the
purified DNA at 4C for short-term use or aliquot the
DNA and store at –20C for long-term storage.
To quantitate yield of your DNA, use UV absorbance or one
of the Quant-iT™ DNA Assay Kits. For more information,
see page 12.
17
Troubleshooting
Introduction
Problem
Low DNA yield
Refer to the table below to troubleshoot problems that you
may encounter when purifying genomic DNA with the kit.
Cause
Incomplete lysis
Solution
 Decrease the amount of
starting material used.
 Be sure to add Proteinase K
during lysis.
 Make sure that the tissue is
completely immersed in the
Lysis Buffer.
 Increase the length of
incubation at 55C.
 For mini quantities of tissue,
homogenize tissue before
lysis.
Poor quality of
starting material
Be sure to use fresh sample and
process immediately after
collection or freeze the sample at
–80C or in liquid nitrogen. The
yield and quality of DNA
isolated depends on the type
and age of the starting material.
Insufficient amount of Vortex the tube containing the
ChargeSwitch®
ChargeSwitch® Magnetic Beads
Magnetic Beads added to fully resuspend the beads in
solution before adding them to
your sample.
Pellet of beads
disturbed or lost
during binding or
washing steps
 Keep the sample in the
MagnaRack™ when removing
supernatant during the
binding or washing steps.
 Remove the supernatant
without disturbing the pellet
of beads by angling the
pipette tip away from the
pellet.
Continued on next page
18
Troubleshooting, Continued
Problem
Low DNA yield,
continued
Cause
Incorrect elution
conditions
Solution
 After adding ChargeSwitch®
Elution Buffer (E5) to the
sample, pipet up and down to
resuspend the magnetic beads
before incubation.
 Incubate the sample at 55C to
improve the yield.
 Do not use water to elute
DNA. Use Elution Buffer (E5)
or TE, pH 8.5.
No DNA recovered
Incomplete
dissociation of DNA
from the
ChargeSwitch®
Magnetic Beads
 Perform additional mixing of
the suspension of beads (by
pipetting up and down).
Water used for elution
Do not use water for elution.
The elution buffer must have a
pH = 8.5–9.0 or the DNA will
remain bound to the
ChargeSwitch® Magnetic Beads.
Use ChargeSwitch® Elution
Buffer (E5) or TE, pH 8.5.
ChargeSwitch®
Magnetic Beads stored
or handled
improperly
 Store beads at room
temperature. Do not freeze the
beads as they will become
irreparably damaged.
 Elute DNA at 65C.
 Make sure that the beads are
in solution at all times and do
not become dried. Beads that
have dried out are nonfunctional.
Eluate containing
DNA is discolored
Magnetic pellet
disturbed during
elution
Repeat the elution step (Eluting
DNA, Steps 4–5, page 11 or
page 17).
Continued on next page
19
Troubleshooting, Continued
Problem
Cause
Solution
RNA contamination
Forgot to add
RNase A
Perform RNase A digestion
prior to binding the DNA to the
magnetic beads.
DNA is sheared or
degraded
Lysate mixed too
vigorously or small
pipette tips used
during mixing
 Use a 1 mL pipette tip set to
900 μL to mix the sample.
Bubbles formed
during mixing steps
Make sure that the pipette tip is
submerged in the solution
during mixing.
DNA repeatedly
frozen and thawed
Aliquot DNA and store at 4C or
–20C. Avoid repeated freezing
and thawing.
DNA contaminated
with DNases
Maintain a sterile environment
while working (i.e. wear gloves
and use DNase-free reagents).
20
 Pipet up and down gently to
mix.
Appendix
Accessory Products
The table below lists additional products available from
Invitrogen that may be used with the ChargeSwitch® gDNA
Tissue Kits. In addition, the table lists a selection of
ChargeSwitch® gDNA Kits that are available for purification
of genomic DNA from other sources. For more information
about these and other ChargeSwitch® gDNA Kits, visit
www.invitrogen.com or contact Technical Support (page 22).
Additional
Products
Product
MagnaRack™
Amount
Catalog no.
1 rack
CS15000
®
50 purifications
CS11000
®
50 purifications
CS11040
®
ChargeSwitch gDNA 50 μL Sheep
Blood Kit
50 purifications
CS11300
ChargeSwitch® gDNA Mini Bacteria Kit
50 purifications
CS11301
ChargeSwitch gDNA Normalized
Buccal Cell Kit
50 purifications
CS11020
ChargeSwitch® gDNA Buccal Cell Kit
50 purifications
CS11021
ChargeSwitch Forensic DNA
Purification Kit
100 purifications
CS11200
Quant-iT™ DNA Assay Kit, High
Sensitivity
1000 assays
Q33120
Quant-iT™ DNA Assay Kit, Broad-Range 1000 assays
Q33130
ChargeSwitch gDNA 100 μL Blood Kit
ChargeSwitch gDNA 1 mL Serum Kit
®
®
E-Gel®
Agarose Gels
and DNA
Ladders
E-Gel® Agarose Gels are bufferless, pre-cast agarose gels
designed for fast, convenient electrophoresis of DNA
samples. E-Gel® agarose gels are available in different
agarose percentages and well formats. In addition, a large
variety of DNA ladders is available from Invitrogen for
sizing DNA. For more information about these products, see
www.invitrogen.com or contact Technical Support (page 22).
21
Technical Support
World Wide
Web
Contact Us
Visit the Invitrogen website at www.invitrogen.com for:

Technical resources, including manuals, vector maps
and sequences, application notes, SDSs, FAQs,
formulations, citations, handbooks, etc.

Complete technical support contact information

Access to the Invitrogen Online Catalog

Additional product information and special offers
For more information or technical assistance, call, write, fax,
or email. Additional international offices are listed on our
Web page (www.invitrogen.com).
Corporate Headquarters:
European Headquarters:
5791 Van Allen Way
Carlsbad, CA 92008 USA
Tel: 1 760 603 7200
Tel (Toll Free): 1 800 955 6288
Fax: 1 760 602 6500
E-mail:
[email protected]
Inchinnan Business Park
3 Fountain Drive
Paisley PA4 9RF, UK
Tel: +44 (0) 141 814 6100
Tech Fax: +44 (0) 141 814 6117
E-mail:
[email protected]
Continued on next page
22
Technical Support, Continued
SDS Requests
SDSs (Safety Data Sheets) are available on our website at
www.invitrogen.com/sds.
Limited
Warranty
Invitrogen is committed to providing our customers with
high-quality goods and services. Our goal is to ensure that
every customer is 100% satisfied with our products and our
service. If you should have any questions or concerns about
an Invitrogen product or service, contact our Technical
Support Representatives.
Invitrogen warrants that all of its products will perform
according to specifications stated on the certificate of
analysis. The company will replace, free of charge, any
product that does not meet those specifications. This
warranty limits Invitrogen Corporation’s liability only to the
cost of the product. No warranty is granted for products
beyond their listed expiration date. No warranty is
applicable unless all product components are stored in
accordance with instructions. Invitrogen reserves the right to
select the method(s) used to analyze a product unless
Invitrogen agrees to a specified method in writing prior to
acceptance of the order.
Invitrogen makes every effort to ensure the accuracy of its
publications, but realizes that the occasional typographical
or other error is inevitable. Therefore Invitrogen makes no
warranty of any kind regarding the contents of any
publications or documentation. If you discover an error in
any of our publications, please report it to our Technical
Support Representatives.
Invitrogen assumes no responsibility or liability for any
special, incidental, indirect or consequential loss or
damage whatsoever. The above limited warranty is sole
and exclusive. No other warranty is made, whether
expressed or implied, including any warranty of
merchantability or fitness for a particular purpose.
23
Purchaser Notification
Limited Use
Label License
No. 5:
Invitrogen
Technology
The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product
and components of the product in research conducted by the
buyer (whether the buyer is an academic or for-profit entity).
The buyer cannot sell or otherwise transfer (a) this product
(b) its components or (c) materials made using this product or
its components to a third party or otherwise use this product
or its components or materials made using this product or its
components for Commercial Purposes. The buyer may
transfer information or materials made through the use of
this product to a scientific collaborator, provided that such
transfer is not for any Commercial Purpose, and that such
collaborator agrees in writing (a) not to transfer such
materials to any third party, and (b) to use such transferred
materials and/or information solely for research and not for
Commercial Purposes. Commercial Purposes means any
activity by a party for consideration and may include, but is
not limited to: (1) use of the product or its components in
manufacturing; (2) use of the product or its components to
provide a service, information, or data; (3) use of the product
or its components for therapeutic, diagnostic or prophylactic
purposes; or (4) resale of the product or its components,
whether or not such product or its components are resold for
use in research. For products that are subject to multiple
limited use label licenses, the terms of the most restrictive
limited use label license shall control. Life Technologies
Corporation will not assert a claim against the buyer of
infringement of patents owned or controlled by Life
Technologies Corporation which cover this product based
upon the manufacture, use or sale of a therapeutic, clinical
diagnostic, vaccine or prophylactic product developed in
research by the buyer in which this product or its
components was employed, provided that neither this
product nor any of its components was used in the
manufacture of such product. If the purchaser is not willing
to accept the limitations of this limited use statement, Life
Technologies is willing to accept return of the product with a
full refund. For information about purchasing a license to use
this product or the technology embedded in it for any use
other than for research use please contact Out Licensing, Life
Technologies, 5791 Van Allen Way, Carlsbad, California
92008 ; Phone (760) 603-7200 or email: [email protected]
©2010 Life Technologies Corporation. All rights reserved.
24
Notes
25
Notes
26
Corporate Headquarters
5791 Van Allen Way
Carlsbad, CA 92008
T: 1 760 603 7200
F: 1 760 602 6500
E: [email protected]
For country-specific contact information visit our web site at www.invitrogen.com
User Manual
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement