320-022121 Expressions10.3_ACD

a newsletter for gene cloning, expression, and analysis

volume 10 issue 3 may 2003

6 Accurate normalization of real-time PCR

8 Obtain fully intact clones

13 Reported success with the FreeStyle™ System

19 Increased in vitro expression yields

Hungry for a convenient array labeling system? SuperScript™ Indirect cDNA Labeling System has all the right ingredients

new product

2

SuperScript™ Indirect cDNA Labeling System

Convenient microarray labeling with SuperScript™ Indirect cDNA Labeling System

T

he SuperScript™ Indirect cDNA Labeling System simplifies target labeling for microarrays by providing an optimized kit based on the most widely adopted protocols for array label-

ing. It combines increased cDNA yields with greater signal intensity, improving the sensitivity of your microarray experiments no assembly required

figure 2 - cDNA yield comparison using SuperScript™ III vs. SuperScript™ II RTs

Using a lab or publicly available protocol for indirect array labeling means having to source and optimize your reagents before

4500

use. This is time consuming and inconven-

4000

ient. The SuperScript™ Indirect cDNA

3500

SuperScript™ III, 800U

cDNA (pmol)

SuperScript™ II, 400U

Labeling System includes all the necessary reagents (except for fluorescent dyes) for microarray target labeling in one optimized

SuperScript™ II, 800U

3000 2500 2000 1500

kit. There’s no need to obtain, assemble, and

1000

optimize reagents from various vendors, sav-

500

ing you hours of time. The SuperScript™

0

1

Indirect cDNA Labeling System is based on

2

4

6

Incubation time (hour)

the most widely used protocols for indirect

cDNA was synthesized with different amounts of SuperScript™ III and SuperScript™ II RTs. The reaction mixtures included 1X first-strand buffer, 5 mM DTT, 10 µg of total RNA, 2 µg oligo(dT)20, 40 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, 0.5 mM dNTP with amino-allyl and aminohexyl replacement, and 1 µCi [α-32P]dCTP. Five microliters of the reaction was taken out and TCA-precipitated. CPM were counted.

cDNA labeling, so you won’t have to adjust to new methods. These conveniences come at no extra cost (figure 1), enabling you to maintain a cost-effective approach to your microarray studies.

increased yield with SuperScript™ III

experiment. The SuperScript™ Indirect

cDNA of current methods employing

Poor cDNA yields can significantly reduce

System uses an optimal amount—800 U of

SuperScript™ II RT (figure 2). You’ll obtain

the sensitivity and reproducibility of your

SuperScript™ III RT—that yields twice the

labeled targets that are truly representative of the expressed transcripts in your sample,

figure 1 - comparison of using self-assembled reagents with SuperScript™ II RT vs. the SuperScript™ Indirect cDNA Labeling System

allowing you to monitor differentially expressed genes with greater sensitivity. With a higher thermostability and much longer half-life (220 minutes at 50°C) than SuperScript™ II RT, SuperScript™ III RT

SuperScript™ Indirect cDNA Labeling System Self-assembled reagents with SuperScript™ II RT

allows you to extend your cDNA synthesis reactions to generate significant yields when starting material is limited (figure 2). Successful microarray hybridizations can be

Performance

Convenience

Cost

performed using as little as five microgams of total RNA (figure 3, page 3). continued on page 3

Expressions 10.3

1 800 955 6288

3 SuperScript™ Indirect cDNA Labeling System

continued from page 2

intense signal

Labeling System takes it one step further. It

everything you need

Most indirect labeling kits use an amino-allyl

uses two modified nucleotides during this

The SuperScript™ Indirect cDNA Labeling

modified nucleotide during first-strand cDNA

stage, increasing the number of available

System comes with all the necessary

synthesis. This modified nucleotide can be

nucleotides that can be post-labeled. This

reagents to effectively produce labeled tar-

effectively “post-labeled” with an N-hydroxy-

unique amino-allyl/aminohexyl (AA/AH)

gets for microarray hybridization (except

succinimide (NHS) ester form of Cy3™/Cy5™

nucleotide mixture increases signal intensity

fluorescent dyes) (table 1). All reagents are

or a range of other commercially available

four-fold over the self-assembled methods

tested for functionality to ensure repro-

(figure 4), enabling greater sensitivity.

ducible fluorescent labeling.

dyes. The

SuperScript™

Indirect cDNA

figure 3 - microarray hybridization using the SuperScript™ Indirect cDNA Labeling System and pooled total RNA

Array (MWG Human Starter) was hybridized overnight with Cy3™-labeled cDNA prepared from 5 micrograms of pooled human total RNA using the SuperScript™ Indirect cDNA Labeling System. The image was acquired using Axon's GenePix® 4000B microarray scanner.

5 µg

figure 4 - greater signal intensity and signal-to-noise ratio with the SuperScript™ Indirect System compared to self-assembled method

table 1 - SuperScript™ Indirect cDNA Labeling System components SuperScript™ III RT

DMSO

Anchored Oligo(dT)20 (2.5 µg/µl)

2X Coupling Buffer

Random Primers

3 M NaAc, pH 5.2

Control RNA Ladder (0.5 µg/µl)

Glycogen (20 mg/ml)

5X First-strand Buffer

S.N.A.P™ Columns

0.1 M DTT

Collection Tubes

10 mM dNTP Mix

Loading Buffer

RNaseOUT™ (40 U/µl) Recombinant Ribonuclease Inhibitor

Wash Buffer

DEPC-water

Amber Collection Tubes

improve your results Target labeling is an integral step in obtaining

A. Signal intensity

B. Signal-to-noise ratio

5000

20

Mean signal-to-noise ratio

Mean Signal (RFUs)

4000

3000

2000

1000

accurate results during microarray experiments. Don’t compromise on sensitivity. The SuperScript™ Indirect cDNA Labeling System

15

provides a convenient means to increase the sensitivity in your experiments, enabling you

10

to obtain better results. Call and order today. 5

Product 0

Quantity

Cat. no.

0


Self-assembled reagents using SuperScript™ II RT


Self-assembled reagents using SuperScript™ II RT

Analysis of arrays (MWG Human Starter) hybridized with Cy3™-labeled cDNA prepared using the SuperScript™ Indirect cDNA Labeling System and common protocol using self-assembled reagents with SuperScript™ II RT. Triplicate labeling reactions were performed and hybridized to array. Graph 4A shows the mean signal intensity of all positive genes for the average of the triplicate reactions. Graph 4B shows the mean signal-to-noise ratios.

SuperScript™ Indirect cDNA Labeling System 10 rxns L1014-01 SuperScript™ Indirect cDNA Labeling System 30 rxns L1014-02

These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or web site, www.invitrogen.com). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.

www.invitrogen.com

Expressions 10.3

new product

4

E-Gel® 48 Pre-cast Agarose Gels

Easy-to-use, 48-well, pre-cast 4% agarose gels increase throughput

S

top wrestling with traditional 4% agarose gels when separating low molecular weight DNA fragments and PCR samples. The new E-Gel® 48 pre-cast agarose gel is a 48-well

medium-throughput, ready-to-use 4% gel that turns this struggle into a Plug & Play operation. easy-to-use 4% pre-cast gel

includes two rows, each containing 24 sam-

results. You’ll always achieve clear band res-

4% agarose gels are notoriously difficult to

ple and 2 molecular weight marker wells,

olution (figure 2) and distinct band separa-

work with—they are hard to pour, difficult to

with a 3.2-cm run length. The well spacing

tion (table 1).

handle, and frequently crack—and they tend

allows loading with an 8- or 12-multichan-

to cloud over and obscure results. The new

nel pipettor. For your convenience, fully

easy-to-use

E-Gel®

48 gels are pre-cast 4%

automated robotic loading is also possible.

agarose gels that get you the medium-

You’ll quickly load samples and be on your

throughput separation results you want with-

way to analyzing results.

figure 2 - clear resolution on the E-Gel® 48 4% agarose gel

out the hassle. Each self-contained E-Gel® 48 gel is a complete electrophoresis system that includes 4% high-resolution agarose, ethidi-

table 1 - distinguish between closely sized bands on E-Gel® 48 gels

um bromide, and electrodes in a 48-well dis-

100 bp

Base pair separation between adjacent bands

posable, UV-transparent cassette. There are

Band size

no gels to pour, buffers to prepare, or stain-

5 bp - 40 bp

ing and destaining procedures to follow, sav-

40 bp - 80 bp

10 bp

ing you hours of time. You’ll effortlessly sep-

80 bp - 175 bp

20 bp

arate small DNA fragments from 10-400 bp.

175 bp - 300 bp

50 bp

300 bp - 600 bp

100 bp

10 bp

5 bp

Various samples, including DNA ladders, dsRNA, and PCR products, ranging in size from 10-400 bp were run on an E-Gel® 48 gel.

designed for fast analysis Using E-Gel® 48 gels, you’ll analyze 48 sam-

Plug & Play bases

simplified separation

ples in just 20 minutes. The gel design

E-Gel® 48 gels run in the E-Gel® 96 mother

The easiest way to separate nucleic acid

and daughter base combinations (figure 1).

fragments between 10 and 400 bp is with

Each base is a gel base and power supply

E-Gel® 48 4% pre-cast gels. You’ll eliminate

all in one and is programmable for the 20-

the tedious task of preparing your gels,

minute run time. The mother base contains

increase throughput, and get better results.

a power cord that plugs directly into any

Call and order today.

figure 1 - the E-Gel 48 gel runs in the E-Gel® 96 mother and daughter bases ®

to electrical outlet

standard outlet. Daughter bases connect to E-Gel® 48 gel

the mother base and to each other to create

Product

multi-unit systems. Each base runs inde-

E-Gel® E-Gel® E-Gel® E-Gel®

pendently, shuts off automatically, and features a built-in digital timer, alarm, and lighted display for Plug & Play operation.

E-Gel® 96 mother base

Cat. no.

48 4% agarose gels 8 gels G8008-04 96 mother base 1 base G7100-01 96 daughter base 1 base G7200-01 Low Range Quantitative DNA Ladder 100 apps 12373-031

clear resolution

E-Gel® 96 daughter bases

The E-Gel® 48 gels are made with the highest resolution agarose so you never have to worry about poor gel quality ruining your

Expressions 10.3

Quantity

1 800 955 6288

These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or web site, www.invitrogen.com). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.

new product

5

PCR Cloning System with Gateway® Technology

Expand your research options with the new PCR Cloning System with Gateway Technology ®

T

he PCR Cloning System with Gateway® Technology provides rapid and efficient construction of Gateway® entry clones. Now this popular method offers a new Gateway® cloning

vector for compatibility with a wider variety of Gateway® destination vectors. unlimited expression options Gateway® Technology enables you to quickly

figure 1 - use recombination to enter the Gateway® Technology

access multiple systems for comprehensive att B

protein characterization and analysis. The

BP Reaction

first step in using the Gateway® system is to

att B

att P

att P

gene

ccdB

attB-flanked PCR product or expression clone

donor vector

att L

att L gene

BP Clonase™

entry clone

recombine your gene of interest with a donor vector to create an entry clone. Then,

att L

LR Reaction

simply recombine the entry clone with a

att L

att R

att R

gene

ccdB

entry clone

Gateway® destination vector to create an

att B

att B gene

LR Clonase™

destination vector

expression clone

expression clone (figure 1). In the PCR In the BP reaction, the gene is transferred into an entry clone. This entry clone can then be used in the LR reaction to create an expression clone.

Cloning Kit with Gateway® Technology and the

pDONR™221

vector, you create

kanamycin-resistant entry clones for use with ampicillin-resistant destination vectors.

and universal M13 sequencing sites (figure 2).

convert your PCR products into entry

Due to selection issues, it is difficult to use

The pUC origin increases plasmid yields of

clones, including the pDONR™/Zeo vector,

these entry clones with kanamycin-resistant

the resultant entry clones, saving you time

BP Clonase™ enzyme mix, Library

destination vectors, such as those used by

and resources. M13 sequencing sites allow

Efficiency™ DH5α™ Chemically Competent

many plant systems. To overcome this limi-

you to easily sequence your entry clones

Cells, buffers, controls, and Zeocin™ selec-

tation, use the pDONR™/Zeo vector that

using universal primers and standard proto-

tion agent. The reagents work together so

confers Zeocin™ resistance to shuttle your

cols. In addition, the Zeocin™ marker is

you achieve optimal results.

gene into even more destination vectors.

potent, which allows you to effectively

high yields, easy sequencing, and effective selection

select Gateway® entry clones.

The pDONR™/Zeo donor vector, as with pDONR™221, has a pUC origin of replication

Use the pDONR™/Zeo vector to rapidly con-

easily convert PCR products to the Gateway® Technology Using the

pDONR™/Zeo

vector to access

Gateway® Technology is straightforward. figure 2 - pDONR™/Zeo vector

M13 forward

M13 reverse

interest. Then incubate the PCR product with

pDONR™/Zeo

and BP

Clonase™

enzyme

mix for one hour. Transform and plate on pU C o r i

pDONR™/Zeo

vert your PCR fragment into an entry clone. Then use the power of Gateway® Technology to access the expression possibilities you need. Call and order today.

Simply design PCR primers with an attB site* and use these to amplify your gene of

ccdB Cm R a ttP attP

start converting today

LB medium with Zeocin™ reagent.

4.7 kb

Product PCR Cloning System with with pDONR™/Zeo pDONR™/Zeo vector

Quantity Gateway® 20 rxns 6 µg

Cat. no.

Technology 12535-027 12535-035

* For more information on primer design, visit the on-line Gateway® Technology seminar at www.invitrogen.com.

convenient cloning in a complete kit The PCR Cloning System with Gateway® Zeocin™

Technology provides everything you need to


www.invitrogen.com

Expressions 10.3

new product

6

Certified LUX™ Primer Sets for Housekeeping Genes

Get accurate real-time PCR normalization

C

ertified LUX™ Primer Sets for Housekeeping Genes provide the tools you need to get accurate normalization of your real-time PCR experiments. With 32 pre-made primer

sets optimized for a wide range of internal control genes with varying expression levels, you’ll match the expression of your target gene, secure the accurate results you need, and get the ready-to-use convenience you want. the right expression level

high performance in every experiment.

tom-designed, target-specific LUX™

Reliable internal standards are critical for

Figure 1 (page 7) demonstrates the precise

Fluorogenic Primers. Each set is convenient-

achieving accurate results in real-time PCR.

amplification you can achieve for genes that

ly provided in 100 µl of 50 µM solution with

Ideally, the internal control should be

express at low, medium, and high levels.

your choice of FAM or JOE label for easy

expressed at roughly the same level as the RNA under study (1). Yet many laboratories use a single housekeeping gene for normalization, despite potential quantification errors introduced as a result of mismatched

the perfect complement to custom-designed LUX™ Primers Certified LUX™ Primer Sets for Housekeeping Genes are designed for easy use with cus-

addition to your PCR. Design your custom LUX™ Primer set for your target gene, choose a Certified LUX™ Primer Set that matches your target expression level, and amplify. It’s that easy.

expression levels between the internal control and gene of interest. Certified LUX™

table 1 - summary of the relative expression levels of various genes compared to 18S rRNA

Primer Sets for Housekeeping Genes provide GenBank accession no.

Relative expression

18S rRNA

X03205

++++

hBETA-ACTIN

NM_001101

+++

hATPSase

NM_001686

sion levels between your target and internal

hB2M

control gene, and gives you the accurate

hGAPDH

quantification you need (2).

hPGK1 hPPIA

optimized for high performance

hRPL4

Certified LUX™ Primer Sets for Housekeeping

GenBank accession no.

Relative expression

18S rRNA

X03205

++++

mBETA-ACTIN

NM_007393

+++

+++

mB2M

X01838

+++

NM_004048

+++

mEEF1G

AF321126

+++

NM_002046

+++

mGAPDH

NM_008084

+++

NM_000291

+++

mPGK1

NM_008828

+++

NM_021130

+++

mPPIA

NM_008907

+++

NM_000968

+++

mRPL4

NM_022510

+++

hEEF1G

NM_001404

++

mHPRT1

NM_013556

++

Genes are functionally validated and opti-

hHPRT1

NM_000194

++

mSDHA

AF095938

++

mized to provide precise amplification of a

hSDHA

NM_004168

++

mATPSase

NM_016774

+

corresponding housekeeping gene. Each of

hTFRC

NM_003234

++

mGUS

NM_010368

+

hGUS

NM_000181

+

mHMBS

XM_129404

+

the solution to this problem. These premade LUX™ Primer Sets are designed for a wide range of internal control genes with varying expression levels (table 1). This wide selection lets you match the expres-

the 32 sets is expressly designed to discriminate between messages and known pseudogenes/different isoforms, and is validated

Name Human genes

Mouse/Rat genes

hHMBS

NM_000190

+

mTBP

NM_013684

+

hTBP

NM_003194

+

mTFRC*

NM_011638

+

hUBC

NM_021009

+

using a dilution series in a two-step realtime RT-PCR with total HeLa RNA, NIH-3T3 RNA, or Drosophila S2 RNA. The amplifica-

Name

* mouse only

Drosophila genes d18S rRNA

AY037174

++++

dACTIN

NM_079486

+++

tion efficiency is greater than 90%, ensuring continued on page 7

Expressions 10.3

1 800 955 6288

7 Certified LUX™ Primer Sets for Housekeeping Genes


the advantages of LUX™

primer. And with multiplexing and melting

wide selection to fit your needs

Using Certified LUX™ Primer Sets, you have

curve analysis capabilities, there’s no need

Certified LUX™ Primer Sets for Housekeeping

to detect with expensive dual-labeled

Genes give you the widest selection of pre-

form: sensitive, specific real-time detection

probes. For more information on the LUX™

made internal controls currently available.

using only a single-labeled fluorogenic

platform, visit www.invitrogen.com/lux.

Primers are available for dozens of human,

the proven advantages of the

LUX™

plat-

mouse/rat, and Drosophila genes, including

primer and a corresponding unlabeled

18S rRNA, GAPDH, and β-actin. With the right Certified LUX™ Primer Set for

figure 1 - amplification with Certified LUX™ Primers for Housekeeping Genes expressing at low, medium, and high levels

Housekeeping Genes, accurate and convenient real-time PCR normalization is only fin-

A. Amplification Plot

45

Low abundance gene mHMBS (Acc. no. NM_129404) Threshold Cycle (C t )

30 ng 3 ng 300 pg 30 pg 3 pg

numbers, call Invitrogen or visit us on-line

y = -3.47(x)+ 37.48 R 2 = 0.996

40

NIH 3T3 cDNA

∆ Rn

gertips away. For a complete list of catalog

B. Standard Curve

at www.invitrogen.com/lux.

35

30

Product

25

Certified

20

05

10

15

20

25

30

35

40

45

15 10 -3

50

10 -2

45

Medium abdundance gene hHPRT1 (Acc. no. NM_000194

-1

10 2

10 1

1

B. Standard Curve y = -3.57(x) + 34.56 R 2 = 0.997

40

Threshold Cycle (C t )

HeLa cDNA 30 ng 3 ng 300 pg 30 pg 3 pg 0.3 pg

∆Rn

10

Starting Quantity (ng of cDNA)

Cycle Number


Quantity

Cat. no

LUX™

Primer Set, FAM-labeled* 100 rxns Certified LUX™ Primer Set, JOE-labeled* 100 rxns Platinum® Quantitative PCR SuperMix-UDG 100 rxns 11730-017 500 rxns 11730-025

35

30

!

25

order on-line

20

* For a complete list of primer sets and 0

5

10

15

20

25

30

35

40

45

15 10

50

-4

10 -3

10 -1

10 1

1

10 2


Cycle Number


45

High abundance gene hATPSase (Acc. no. NM_001686 Threshold Cycle (C t )

30 ng 3 ng 300 pg 30 pg 3 pg 0.3 pg

www.invitrogen.com/lux.

y = -3.42(x) + 29.62 R 2 = 0.996

References: 1. Bustin, S.A. (2000) J. of Mol. Endocrinol. 25: 169-93. 2. Vandesompele, et al. (2002) Genome Biology 3:(7) 1-10.

35

30

25


20

0

5

10

15

20

25

30

Cycle Number

35

40

45

50

catalog numbers, please visit

B. Standard Curve

40

HeLa cDNA

∆ Rn

10 -2

15 10 -4

10 -3

10 -2

10 -1

1

10 1

10 2


Panel A: Real-time PCR of 10-fold serial dilutions of HeLa cDNA (hATPSase-high and hHPRT1-medium) or mouse NIH 3T3 cDNA (mHMBS-low) were performed using 200 nM JOE-labeled LUX™ Primer, 200 nM unlabeled primer, Platinum® Quantitative PCR SuperMix-UDG, and ROX Reference Dye. Reactions were incubated for 2 min. at 50°C, then 2 min. at 95°C, followed by 50 cycles of 95°C, 15 sec.; 60°C, 30 sec. using an ABI PRISM® 7700. Panel B: Standard curves showing the starting template amount versus Ct value.

www.invitrogen.com

Expressions 10.3

new product

8

CloneMiner™ cDNA Library Construction Kit

Construct cDNA libraries without restriction enzymes—get clones never before available

T

he CloneMiner™ cDNA Library Construction Kit enables you to construct highly representative cDNA libraries without the risk of restriction enzymes cutting your precious gene.

Now you can isolate fully intact clones from high-quality cDNA libraries. overcome pitfalls of library construction

figure 1 - the CloneMiner™ cDNA Library Construction Kit method

The traditional method of cDNA library con-

mRNA

(A)n

mRNA

(A)n (T)n

struction has many inherent pitfalls, includ-

1. Start with mRNA.

ing: 1) poor cDNA synthesis, 2) inefficient ligations, and 3) nicking or cutting your clone during restriction enzyme digestion.

2. Anneal oligo dT-attB2-Biotin primer. B

Any one of these may lead you to lose your clone of interest, especially if the gene

B attB2-Biotin

occurs in low abundance. If you do get your clone, it may be digested by a restriction

3. Synthesize first- and second-strand cDNA using SuperScript™ III RT.

4. Ligate attB1adapter. cDNA

enzyme or lost in subsequent subcloning

attB2-Biotin

attB1

steps due to inefficient ligation reactions.

B

The CloneMiner™ cDNA Library Construction cDNA

Kit provides an entirely new, more efficient attB1

method for generating libraries without the

B attB2-Biotin

ccdB attP1

attP2

Donor Vector pDONR™222

limitations of restriction enzyme cloning. You’ll uncover clones never obtained before

Knr

by traditional methods.

5. Recombine adapted cDNA with pDONR™222 vector in the presence of BP Clonase™ enzyme mix to create the entry library.

top-notch cDNA library construction The new CloneMiner™ Kit takes advantage of two leading-edge technologies to enable

cDNA

you to obtain the highest percentage of full-

attL1

length genes and eliminate restriction-diges-

ccdB

attR1

tion bias from your library construction

B attR2-Biotin

attL2

Entry Library

(figure 1): • SuperScript™ II reverse transcription tech-

Knr Legend:

nology delivers a very high percentage of full-length cDNA from your starting mRNA material. SuperScript™ II reverse transcriptase (RT) offers reduced RNase H activity, dramatically minimizing RNA

Gateway® attB site

B

Biotin tag

6. Resulting entry clones have attL sites; resulting byproduct is the free ccdB gene.

7. Transform E. coli and select for Kn resistant colonies.

degradation during first-strand synthesis. continued on page 9

Expressions 10.3

1 800 955 6288

9 CloneMiner™ cDNA Library Construction Kit


• Gateway® Technology, a well-characterized

eliminates this risk. No clone is ever

site-specific recombination system, medi-

exposed to a restriction enzyme, providing

ates actual library construction. This

you with optimal clone representation and

allows transfer of your cDNA fragments

100% fully intact cDNA (figure 2).

into a Gateway® donor vector to create an entry library. DNA is then recombined into a variety of expression vectors via Gateway® Technology. The reaction maintains orientation and reading frame, so there are no extra revalidation steps needed. The use of restriction enzymes and ligase is effectively eliminated. You’ll construct a high-quality library for efficient

choose your screening method CloneMiner™

Product

Quantity

Cat. no.

CloneMiner™ cDNA Library Construction Kit 5 rxns* 18249-029

* Sufficient reagents for constructing 5 cDNA libraries.

cDNA libraries can be

screened using any number of techniques to isolate specific sequences of DNA,

These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or web site, www.invitrogen.com). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use.

including traditional methods like probe screening or PCR amplification. Alternatively, an entire library may be transferred into an expression vector for

!

Easy transfer to other expression systems

expression cloning and functional analysis.

Using Gateway® Technology, you can easi-

Since the CloneMiner™ cDNA Library

ly transfer your gene of interest into other

obtain fully intact clones

Construction Kit incorporates Gateway®

systems, including mammalian, insect,

Traditional cloning methods require the use

Technology, you have immediate access to

yeast, or E. coli for additional studies—

of restriction enzymes to adapt the ends of

a broad range of expression systems.

without subcloning (figure 3). A simple

everything you need

sites present in every Gateway® entry and

To make constructing your library conven-

destination vector allows you to take

ient, the CloneMiner™ cDNA Library

advantage of a broad range of expression

Construction Kit includes everything you

options. To learn more about Gateway®

need (table 1). There’s no need to search

Technology, visit www.invitrogen.com.

and comprehensive analysis.

your cDNA to fit into a cloning vector—running the risk that these same enzymes may cut your gene into multiple segments. By using Gateway® Technology in place of restriction enzymes, the CloneMiner™ Kit figure 2 - the CloneMiner™ Kit provides the highest percentage of fully intact cDNA clones

recombination reaction between the att

for additional reagents. All you need to supply is your starting material.

figure 3 - Gateway® Technology overview DNA fragments from:

table 1 - components in the CloneMiner™ cDNA Library Construction Kit

100

PCR Cloning Restriction Digests

90

% fully intact clones

E. coli

SuperScript™ II Reverse Transcriptase

80

cDNA Libraries

Your Vector Gene

Gene

Gateway® vector

70 60

pDONR™ 232

50

Entry Clone

Yeast Gene

Gene

Two-Hybrid Gene

Reagents for cDNA library construction

40

Insect

30

ElectroMAX™ DH10B™ T1 Phage-Resistant competent cells

20 10

Mammalian Gene

Gene In Vitro Gene

0

CloneMiner™ Kit

Two restriction enzymes (6-mer and 8-mer)

Two restriction enzymes (6-mers)

CloneMiner™ Technology offers the highest percentage of fully intact clones compared to traditional methods. Percentage is calculated based on the statistical frequency a given enzyme will cut a 1.5 kb insert.

superior technology yields highest quality libraries

1. Create a Gateway® entry library 2. Mix with a Gateway® destination vector of your choice.

Using the CloneMiner™ cDNA Library Construction Kit, you’ll detect rare or novel genes never before available. Find your gene by calling Invitrogen and ordering today.

www.invitrogen.com

Expressions 10.3

product review

10

TOPO® Cloning Technology

TOPO Cloning Technology–easier, faster, and more efficient than restriction enzyme cutback and ligation ®

I

nvitrogen’s TOPO® Cloning Technology is the most effective method available for cloning PCR products and other DNA molecules. It yields up to 99% recombinants via a simple

5-minute, bench-top ligation. If you are using other methods of PCR cloning, switching to TOPO® Cloning will improve your results and save you time. the technology behind TOPO® Cloning

TOPO® Cloning works better than ligase

• In a TOPO® Cloning reaction, only two molecules must come into contact for liga-

Cloning you’ll get your clones

tion to occur—the TOPO® vector and the

faster and more successful. It enables 5-

fast, and on your first try. Topoisomerase is

insert. This is more kinetically favorable

minute, bench-top ligation and yields up to

superior to ligase for several reasons:

than conventional ligation where three

TOPO®

Cloning Technology makes ligations

99% recombinants. The key to

TOPO®

Cloning is the enzyme vaccinia virus DNA topoisomerase I, which functions as both a restriction enzyme and a ligase. Its biological role is to cleave and rejoin DNA during replication. To harness the religating activity of topoisomerase, TOPO® Cloning vectors are provided linearized with topoisomerase

With

TOPO®

molecules—the vector, the insert, and lig• Ligase is often contaminated by nucleases

ase—must interact in the presence of ATP

that nick DNA and vector ends, reducing

for ligation to occur

the number of recombinants • TOPO® Cloning vectors have topoiso-

For these reasons, ligase-mediated cloning

merase I covalently bound to their ends,

results in only 60% cloning efficiency com-

protecting the vector ends from exonucle-

pared to the up to 99% cloning efficiency

ases and maintaining their integrity

achieved with TOPO® Cloning.

I covalently bound to each 3´ phosphate. This enables them to readily ligate DNA

table 1 - restriction site cutback vs. TOPO® Cloning Technology

sequences with compatible ends. After only 5 minutes at room temperature, the ligation

Step

is complete and ready for transformation

Order or prepare

Special primers containing

Add 10 extra bases to each PCR primer

into E. coli.

PCR primers

extra bases are not required

to create restriction sites (6 for the

TOPO® Cloning

Restriction enzyme cutback and ligation

restriction site, 4 for the spacing)

three easy steps TOPO® Cloning requires just three easy steps.

Prepare the vector and

Linearized TOPO® Cloning

1) Digest vector and PCR product

PCR product for ligation

Vectors are ready for direct

1) with restriction enzymes(s)

Simply mix your PCR product and a TOPO®

ligation of unmodified,

2) Gel purify the digested PCR

Cloning vector, wait five minutes, and trans-

unpurified PCR products

1) product using low-melt agarose

form E. coli. With TOPO® Cloning

Obtain ligation reagents

Technology, the additional time, steps, and

All required cloning reagents

Purchase ligase, ATP, and ligation

are included

buffer, E. coli

reagents required for ligase-mediated cloning

Prepare or purchase

TOPO® Cloning Kits include

1) Purchase: 0 hours

are eliminated. This means there are:

competent cells

One Shot® Competent E. coli

2) Prepare: 6 hours

Incubate the ligation

5 minutes

6 to 23 hours

Recombination efficiency

Up to 99%

~ 60%

Total time for cloning

5 minutes

6 to 23 hours

• No ligase or overnight incubations • No need for PCR primers that contain restriction sites* • No post-PCR modifications or gel purifica-

continued on page 11

tion needed**

Expressions 10.3

1 800 955 6288

11 TOPO® Cloning Technology


clone your exact sequence

sequence—without any extras. The restric-

in your PCR products. Unless you know the

tion site cutback method forces you to add

entire sequence of your PCR product before

Cloning Technology allows you to amplify

extra sequence to your primers. This may

amplification, you may unintentionally

your gene of interest with specific primers

cause non-specific amplification and com-

cleave it when you digest its ends. You

that do not contain additional bases. This

promises the final sequence of your PCR

avoid these potential pitfalls when you use

allows you to directly clone unmodified PCR

product. In addition, the restriction sites you

TOPO® Cloning.

products that represent your exact

add to your primers may already be present

Unlike restriction enzyme cloning,

TOPO®

save time with TOPO® Cloning In addition to the limitations mentioned

table 2 - TOPO® Cloning products

above, restriction site cutback takes more time, requires additional steps, and is less

For cloning Taq-amplified PCR products

Cat. no.

effective than TOPO® Cloning. Table 1 (page 10) details some of the extra steps required

TOPO TA Cloning® Kit (with pCR®2.1-TOPO® vector) with One Shot® TOP10 Chemically Competent E. coli

K4500-01

in restriction site cutback method and pro-

with One Shot® TOP10F´ Chemically Competent E. coli

K4550-01

vides conservative estimates of the time

with One Shot® TOP10 Electrocomp™ E. coli

K4560-01

saved using TOPO® Cloning Technology.

with One Shot® MAX Efficiency® DH5α™-T1R E. coli

K4520-01

start TOPO® Cloning today

TOPO TA Cloning® Kits Dual Promoter (with pCR®II-TOPO® vector) with One Shot® TOP10 Chemically Competent E. coli

K4600-01

with One Shot® TOP10F´ Chemically Competent E. coli

K4650-01


K4660-01


K4620-01

TOPO® Cloning gives you better results, faster. Don’t settle for any other PCR cloning method. TOPO® Cloning Kits are available for quick, efficient cloning of Taq-

For cloning blunt-end PCR products (PCR products amplified with proofreading polymerases)

amplified, blunt-end, and long PCR prod-

Zero Blunt® TOPO® Cloning Kits (with pCR®-Blunt II-TOPO® vector)

ucts. Refer to table 2 or for a complete list

with One Shot® TOP10 Chemically Competent E. coli

K2800-20


K2860-20


K2820-20

of products visit the Invitrogen web site (www.invitrogen.com/topo) and order your TOPO® Cloning Kit today.

For cloning long PCR products (3-10 kb) TOPO® XL PCR Cloning Kit (with pCR®-XL-TOPO® vector) with One Shot® TOP10 Electrocomp™ E. coli

K4700-20

with One Shot® TOP10 Chemically Competent E. coli

K4750-20

* The Directional TOPO® Cloning products require the 5´ PCR primer to be designed with CACC at the 5´ end (no modification of the 3´ primer is necessary). ** The TOPO® XL PCR Cloning Kit requires a 15-minute gel purification step.

For cloning and streamlined sequencing of PCR products TOPO TA Cloning® Kit for Sequencing (with pCR®4-TOPO® vector) with One

Shot®

TOP10 Chemically Competent Cells

K4575-01


K4580-01


K4595-01

These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or our web site, www.invitrogen.com). Products referred to herein which are the subject of one or more of U.S. Patent No. 5,910,438 and 6,180,407, and/or corresponding foreign patents, are sold under license from the Université Libre de Bruxelles for research purposes only (Limited Use Label License No. 54: ccdB-Fusion Vectors).

Zero Blunt® TOPO® Cloning Kit for Sequencing (with pCR®4Blunt-TOPO® vector) with One Shot® TOP10 Chemically Competent Cells

K2875-20


K2880-20


K2895-20

www.invitrogen.com

Expressions 10.3

new product

12

SuperScript™ III First-Strand Synthesis System for RT-PCR

Make high cDNA yields easy

T

he SuperScript™ III First-Strand Synthesis System for RT-PCR delivers the superior performance and convenient format you need for your cloning and gene expression analysis

experiments. With higher cDNA yields, increased priming specificity, and a streamlined protocol, successful first-strand cDNA synthesis has never been easier. performance and convenience combined

figure 1 - the SuperScript™ III First-Strand Synthesis System outperforms the competition

The SuperScript™ III First-Strand Synthesis System for RT-PCR combines the high cDNA yields and increased priming specificity of SuperScript™ III Reverse Transcriptase (RT) with the convenience of a comprehensive kit. Whether you’re generating cDNA for cloning or detecting rare messages, the

Total HeLa RNA (ng)

Promega

Stratagene

Applied Biosystems

ImProm-II™ RT System

ProSTAR® First Strand RT-PCR Kit

GeneAmp® Gold RNA PCR Kit

42˚C – 60 min 1 100

42˚C – 12 min 1 100

42˚C – 60 min 1 100 M

M

M

Roche

Invitrogen

First Strand cDNA Synthesis Kit for RT-PCR (AMV)

SuperScript™ III First-Strand Synthesis System for RT-PCR 50˚C – 50 min 1 100

42˚C – 60 min 1 100 M

M

M

FIB 9.4 kb Pol ε 6.8 kb VIN 4.6 kb

SuperScript™ III System provides everything you need for getting high-quality, first-

PP2A 1093 bp

strand cDNA from total or poly(A)+ RNA. And with a new protocol that reduces, or eliminates, certain RT incubation steps, your research will move forward even faster.

the power of SuperScript™ III RT

RT reactions containing 1 or 100 ng of total HeLa RNA were performed with each kit using reagents and conditions specified in each manufacturer’s protocol. Ten percent (2-5 µl) of the resulting cDNA was added to PCR reactions containing 1 unit of Platinum® Taq DNA Polymerase High Fidelity for 35 PCR cycles, 1 min/kb. PCR products were separated on a 1% agarose gel containing 0.4 µg/ml ethidium bromide.

SuperScript™ III RT, included in the SuperScript™ III First-Strand Synthesis System, is an RNase H- point mutant of

long templates (9.4 kb) at low RNA concen-

your path to success

SuperScript™

trations (100 ng), the SuperScript™ III First-

Ensure your success with every RT experi-

half-life and increased thermostability to

Strand System yields more cDNA.

ment. Call Invitrogen and order the

55°C. This robust RT is active for 220 min-

everything you need to succeed

II RT that exhibits a longer

utes at 50°C, giving you higher cDNA yields, greater success with RNA secondary structure, and increased priming specificity with gene-specific primers. Plus, you’ll get full-length transcripts up to 12.3 kb and sensitivity to 1.0 pg total RNA.

The SuperScript™ III First-Strand Synthesis System provides all components necessary for optimal first-strand cDNA synthesis, including 10X RT buffer, oligo(dT)20 primer, random hexamers, dNTP mix, RNaseOUT™ Recombinant Ribonuclease Inhibitor, MgCl2,

the highest yield of the field

DTT, RNase H, DEPC-treated water, and

Figure 1 shows the superior yields and long

total HeLa RNA control with primers. With

cDNA generated by the

SuperScript™

III

First-Strand Synthesis System compared to other first-strand synthesis kits. Even with

Expressions 10.3

1 800 955 6288

everything ready to go, you can start experiments sooner and get results faster.

SuperScript™ III First-Strand Synthesis System for RT-PCR today. Product

Quantity

Cat. no.

SuperScript™

III First-Strand Synthesis System for RT-PCR 50 rxns 18080-051 15 µg 18418-020 Oligo(dT)20 Primer Platinum® Taq DNA Polymerase High Fidelity 100 rxns 11304-011

These products may be subject to one or more Limited Use Label Licenses. Please refer to the Invitrogen catalog or web site for the corresponding Limited Use Label Licenses. All products are for research use only. Caution: Not intended for human or animal diagnostic or therapeutic uses.

product review

13

FreeStyle™ 293 Expression System

Easy, high-yield production of mammalian proteins

J

oin hundreds of scientists and take advantage of the FreeStyle™ 293 Expression System. The complete FreeStyle™ System is the only method that provides rapid, easy, and high-

yield mammalian protein production. you need protein

a complete optimized system. All you need

here’s what researchers are saying

Like many researchers, you need functional

is a eukaryotic expression plasmid contain-

People are talking about how the FreeStyle™

proteins for downstream applications. You

ing your gene of interest. Easy because the

System is successfully solving their protein

need a lot and you need it fast. You spend too

FreeStyle™ system utilizes both suspension

production challenges. Read the boxes to see

much time tediously feeding, passaging, and

culture and serum-free media. FreeStyle™

what they are saying.

transfecting dozens or even hundreds of indi-

293-F cells are pre-adapted to suspension

vidual tissue culture flasks. All just to get

culture, so you can generate as much pro-

enough protein for your experiments. Or you

tein in one easy-to-handle shaker flask as

use yeast, insect cells, or bacteria, even when

you can with four or five awkward T-160

mammalian hosts would be more appropri-

cm2 tissue culture flasks. In addition, the

ate. Suffer no more. Now produce large quan-

GIBCO™ FreeStyle™ 293 Expression Medium

tities of protein in mammalian cells quickly

is serum-free for easier and faster down-

and easily with the FreeStyle™ 293 Expression

stream processing and purification.

Dr. Charlotte Dyring, at Pharmexa A/S in Denmark uses the system to express antigenic proteins. She states, “We have achieved very high levels of protein expression for large-scale transient transfections. The cells are growing

System. Join researchers around the world who have solved their protein production

get results in just two weeks

well and to a rather high cell density. It 25-fold improvement

is of great value to us that transfection

A researcher at a major New England

can take place directly in the growth

powerful protein production

pharmaceutical company reported: “I

medium in suspension. We produce

The FreeStyle™ 293 Expression System has

saw a 25-fold improvement in produc-

enough protein to make initial purifica-

three powerful components:

tion of a secreted protein when using

tion experiments by simply making

challenges with the FreeStyle™ System.

• FreeStyle™ 293-F cells—for high-level protein expression in serum-free media • 293fectin™ transfection reagent—for trou-

the

FreeStyle™

System compared to a

number of other stable systems. This

thereby speeding up the project several

system is easy to grow and easy to use.”

weeks because we do not have to wait for stable cell lines to be established.”

ble-free high-efficiency transfection in FreeStyle™ 293-F cells

transient transfections in shake flasks

protein workhorse

now it’s your turn

• GIBCO™ FreeStyle™ 293 Expression Medium—an optimized chemically

Dr. Sumit Chanda at The Genomics

Experience success by putting the FreeStyle™

defined, serum-free medium

Institute of the Novartis Research

293 Expression System to work in your lab.

Foundation (GNF) in San Diego, CA is

Order today.

Cells, medium, and transfection reagent

enthusiastic about the FreeStyle™

work together for high-yield protein produc-

System, “It’s a workhorse for protein

Product

tion in your lab.

purification. We have expressed a vari-

FreeStyle™

fast and easy The FreeStyle™ system is the fastest, easiest way to express large amounts of proteins in mammalian cells. Fast because FreeStyle™ is

ety of nuclear and cytoplasmic proteins

Quantity 293 Expression System 1 kit

Cat. no. K9000-01

and all have been functional. This system has been extremely versatile and shows no limitations.”


www.invitrogen.com

Expressions 10.3

questions? answers!

14

Competent Cells for Cloning

Choose the best competent cell for cloning

T

here are many varieties of competent cells available. Choosing the best competent cell for cloning applications is critical to your experiment’s success. Below are answers to

some commonly asked questions regarding competent cells used for cloning.

?

What transformation efficiency do I need for cloning?

table 1 - important genetic markers for cloning

Choose a high-efficiency competent cell for

Genotype

Description

demanding cloning applications such as

tonA

Guards against T1 and T5 phage infection, preventing loss of precious DNA clones

library construction and a lower efficiency

endA1

Cleaner preparations of DNA and better results in downstream applications due to the elimination of non-specific digestion by Endonuclease I

for less demanding applications, such as

recA1

Reduced occurrence of unwanted recombination in cloned DNA

subcloning. Generally, electrocompetent

lacZ∆M15

Blue/white color screening of recombinant clones carrying the alpha fragment of β-galactosidase

mcrA, mcrBC, or mrr

Eliminate restriction of methylated DNA normally recognized as foreign. This genotype is necessary when cloning genomic DNA or methylated cDNA

competent cell as a cost-effective alternative

cells have a higher transformation efficiency (>0.5–1 x 1010 cfu/µg) than chemically competent cells (>1–5 x 109).

?

What are the benefits to using T1 Phage-Resistant (T1R) Competent Cells?

?

What genetic markers are important for cloning?

competent cloning: the choice is yours

A variety of genetic markers exist in strains

For successful cloning experiments, you

T1 Phage-Resistant competent cells carry the

used for cloning. Having these markers

need to choose the right strain. Table 2 lists

tonA marker that protects them from being

present helps ensure successful introduc-

just a fraction of the strains available. Call

infected with T1 phage. This safeguards your

tion, propagation, and analysis of recombi-

800 955 6288 to discuss your needs more

valuable clones, stocks, and libraries from a

nant DNA (table 1).

fully or place your order today.

potentially disastrous T1 infection.

table 2 - a selected number of Invitrogen™ cloning strains Product

Application

Benefits

Efficiency

Quantity

Cat. no.

One Shot® OmniMAX™-T1R Chemically Competent E. coli

All cloning applications

• Versatile genotype • Very high efficiency • T1 Phage-resistant

>5 x 109

20 x 50 µl

C8520-03

One Shot® Mach1™-T1R Chemically Competent E. coli

Fast growth for routine cloning

• Mini-preps in 4 hrs. • Colonies in 8 hrs.* • T1 Phage-resistant

>1 x 109

20 x 50 µl

C8620-03

Subcloning Efficiency® DH5α™ Chemically Competent E. coli

Economical subcloning

• Great value

>1 x 106

4 x 500 µl

18265-017

ElectroMAX™ DH10B™-T1R E. coli

The most demanding applications

• Highest transformation efficiency • T1 Phage-resistant

>1 x 1010

5 x 100 µl

12033-015

* When transformed with vectors containing the ampicillin resistance marker.

Expressions 10.3

1 800 955 6288

product update

15

ViraPower™ Lentiviral Expression System

Expanded options for stable gene expression in non-dividing mammalian cells

T

he ViraPower™ Lentiviral Expression System revolutionizes gene delivery and long-term expression in non-dividing mammalian cells. Two new vectors expand your ViraPower™

Lentiviral System options for powerful mammalian cell expression and in vivo animal applications. powerful expression Many expression experiments fall short of

figure 1 - pLenti-DEST™ Gateway® vectors

CmR

attR1

could be due to inefficient cell transfection

ccdB

V

attR2

ψ

RR

EM

E

pLenti4/ V5-DEST™ pU

Ampicillin

CmR

attR1

any cell type. This powerful system now

The new ViraPower™ Lentiviral vectors

RR

0

EM

E

gene of interest when the CMV promoter is

ψ

∆U3 /3’ LTR

pLenti6/UbC/ V5-DEST™ i

Ampicillin

pA 40 SV

or

now select cells that stably express your gene of interest with the pLenti4/V5-DEST™ vector that provides Zeocin™ resistance. In addition, this vector (figure 1) offers the following features: • V5 epitope tag for easy detection of your recombinant protein • att sites for use with Gateway® Technology • Lentiviral vector components for high packaging efficiency • CMV promoter for high-level expression

expand your applications, enabling you to make double stable cell lines, express your

PSV4

idin stic Bla

P RSV/5’ LTR

Stop

C

provide you with greater choices, you can

V5 epitope

pU

conferred resistance to Blasticidin only. To

attR2

7

The original ViraPower™ Lentiviral vectors

ccdB

P ubc

offers an expanded selection of vectors, so

dual selection now possible

resistance gene, respectively (figure 1).

expanded expression options

pA 40 SV

i

Lentiviral Expression System enables you

you have even more options than before.

and Zeocin™ resistance gene are replaced

C

or

even non-dividing cells, the ViraPower™ to deliver your gene of interest to virtually

DEST™ vectors except that the CMV promoter with the UbC promoter and the Blasticidin

Lentiviral Expression System. With its broad tropism and the ability to integrate into

all the same features as the pLenti4/V5-

∆U3 /3’ LTR

P RSV/5’ LTR

ing experiments using the

ViraPower™

Gateway® vector allows you to take advantage of this unique promoter. This vector has

0

™ ocin Ze

you can perform these previously challeng-

Stop

7

or the inability of traditional retroviral sys-

V5 epitope

PSV4

P CM

tems to transduce non-dividing cells. Now

ral context for sustained in vivo gene expression (2). Now the pLenti6/UbC/V5-DEST™

the desired results when genes are not efficiently delivered to cells. This problem

and has been successfully used in a lentivi-

you can choose Blasticidin or Zeocin™ selection, or use both to easily make the dual stable cell lines your research requires.

human promoter for unique applications

not optimal, and even perform research using in vivo model systems. Order today. Product

Quantity

ViraPower™

Gateway®

Cat. no.

Zeo Lentiviral Expression Kit 1 kit K4980-00 pLenti4/V5-DEST™ Gateway® Vector 6 µg V498-10 ViraPower™ Zeo Lentiviral Support Kit* 20 rxns K4985-00 ViraPower™ Ubc Lentiviral Gateway® Expression Kit 1 kit K4990-00 pLenti6/UbC/V5-DEST™ Gateway® Vector 6 µg V499-10 ViraPower™ Bsd Lentiviral Support Kit* 20 rxns K4970-00

The human Ubiquitin C (UbC) cellular promoter is ubiquitously active in mammalian cells and has been shown to evade the silencing signals that can downregulate the

Additionally, all ViraPower™ Lentiviral vectors

CMV promoter in some cells and tissues (1).

are designed to produce non-replicating viral

The UbC promoter is especially compatible

particles and have many safety features. Now

with the generation of transgenic animals

* ViraPower™ Support Kits contain ViraPower™ Packaging Mix, Lipofectamine™ 2000, and selection agent. The ViraPower™ Packaging Mix is also available separately. References: 1. Yew, et. al. (2001) Mol. Ther. 4: 75-82. 2. Lois, et. al. (2002) Science 295: 868-72.

www.invitrogen.com

Expressions 10.3

product review

16

Protein Standards

Ready-to-use standards for all your protein electrophoresis needs

W

hether you’re monitoring electrophoresis runs, verifying blotting transfer efficiency, looking for the most accurate molecular weight estimations, performing western blot

analysis, or determining pI, Invitrogen offers a protein standard to meet your needs. Each ready-to-use standard offers crisp, clear bands for reliable identification and results. standards for all needs

• Accurate results

nal proteins. To overcome this potential pit-

Protein standards play an integral part in

• A convenient, ready-to-use format

fall, Invitrogen offers two unstained protein

your analysis, providing reliable sizing capa-

standards: the Mark12™ Unstained Standard

accurate protein sizing

bilities and an internal standard for your

For the most accurate molecular weight esti-

electrophoresis runs. To obtain accurate

mations in SDS-PAGE, use an unstained pro-

results, you need to use a standard geared towards your experimental goals. That’s why Invitrogen offers eight different protein standards. Each one is specially designed to meet particular electrophoresis needs and offers:

tein standard. The dye present in the proteins of pre-stained standards provides easy visualization, but it can affect band migration patterns in electrophoresis. This may result in apparent molecular weights that significantly differ from those of their origi-

• Consistent, reliable band resolution

(figure 1A) offers 12 bands from 2.5 kDa to 200 kDa and the BenchMark™ Protein Ladder (figure 1B) which features affinitypurified recombinant proteins in even increments for easy identification. The standard bands are visualized by staining with Coomassie® blue, silver stain, or other protein staining reagents.

figure 1 - ready-to-use standards for protein electrophoresis

Unstained protein standards for accurate molecular weight estimation

A

kDa 200 116.3 97.4 66.3 55.4 36.5 31 21.5 14.4 6 3.5 2.5

Mark12

™

NuPAGE® 4-12% Bis-Tris Gel w/MES, stained with Coomassie® R-250

B

kDa 220 160 120 100 90 80 70 60 50 40

Pre-stained protein standards for easy and clear band identification

C

kDa 185 98

D

kDa 188 98 62

52

49

E

kDa ~190

31

30 25 20

19 17

~85 ~60

15

6

10

3

~40

11

G

H

kDa 188

62 49 38 28

28 17 14

IEF marker for pI determination of protein

kDa

~25 ~20

18 14

100

120 80 60 50 40 30

6.9

4.5 4.2

6 ~10 3

3

7.4

6.0 5.3 5.2

20

~15 6

pI 8.3 8.0 7.8

~120

~50 38

F

Protein standard for western blots

3.5

™

MultiMark

SeeBlue Plus2

BenchMark Pre-Stained*

SeeBlue

MagicMark

IEF Marker 3-10

4-20% Tris-Glycine Gel, stained with Coomassie® R-250

NuPAGE® 4-12% Bis-Tris Gel w/MES SDS Buffer


4-20% TrisGlycine Gel


NuPAGE® Bis-Tris Gel/Nitrocellulose membrane, detected with WesternBreeze® Chemiluminescent Kit

Novex® pH 3-10 IEF Gel

BenchMark

®

®

™

®

™

continued on page 17

Expressions 10.3

1 800 955 6288

17 Protein Standards


efficiently monitor gel runs

estimation. With the MagicMark™ Standard,

for additional “high pI range” or “low pI

Pre-stained protein standards allow you to

you’ll obtain sharp bands and precise molec-

range” markers. Use the IEF Marker 3-10 to

easily monitor the progression of your elec-

ular weight estimations directly on your

produce dependable results on horizontal or

trophoresis run. The proprietary technology

western blots. For added flexibility, you can

vertical IEF gels.

and manufacturing process ensure that pro-

visualize bands to estimate molecular weight

tein bands are evenly stained and consis-

in SDS-PAGE using Coomassie® blue stain.

tently sharp for reliable identification. Bright colors make it possible to analyze bands at a glance. Four pre-stained standards are currently available: • the

MultiMark®

Multi-Colored Protein

All of Invitrogen’s protein standards are

maximize success in western analysis

supplied ready to use straight from the vial.

To increase confidence and accuracy in your

There are no mixing, diluting, or heating

western blot experiments, use the

SeeBlue®

steps, saving you time and effort. In addi-

Pre-Stained and MagicMark™ Western

tion, you’ll eliminate the risk of introducing

Standards together. Simply pre-mix the

errors in your preparation steps.

Standard (figure 1C) offers nine multi-

appropriate amount of SeeBlue® and

colored bands in the range of 4-250 kDa†

MagicMark™ standards and load onto the

for immediate and unambiguous band

gel. You’ll see bright blue-stained SeeBlue®

identification

bands during the electrophoresis run and

• the SeeBlue® Plus2 Pre-Stained Protein

after transfer to the membrane (figure 2A).

Standard (figure 1D) offers eight blue

Following immunodetection of your choice,

bands plus two colored bands in the range

sharp and clean MagicMark™ bands develop

of 4-250 kDa† for easy band identification;

directly on blots (figure 2B). You’ll easily

• the BenchMark™ Pre-Stained Protein

convenient, ready-to-use format

and accurately analyze your target protein.

large selection meets your needs To meet different protein analysis needs, Invitrogen offers eight different protein standards. In addition to individual advantages, all produce sharp, clear bands for accurate analysis and come in a convenient, readyto-use format. Choose the one that best fits your experiment and order today.

Ladder (figure 1E) includes affinity-purified recombinant proteins in nine blue bands and one red band in the range of

Product

figure 2 - combine SeeBlue® Pre-Stained and MagicMark™ Western Standards together

~10-190 kDa* • SeeBlue® Pre-Stained Protein Standard

A

B

(figure 1F) consisting of nine blue bands from 4 to 250 kDa† for producing sharp band resolution.

direct visualization on western blots

A: The SeeBlue®-stained bands on a nitrocellulose membrane after transfer B: MagicMark™ bands detected using chemiluminescence

The MagicMark™ Western Protein Standard (figure 1G) lets you visualize molecular weight bands directly on western blots. Each MagicMark™ band contains an IgG binding

SeeBlue® (5 µl) and MagicMark™ (5 µl) Standards pre-mixed prior to loading on a NuPAGE® Bis-Tris Gel/Nitrocellulose membrane.

Quantity

Cat. no.

Mark12™

Unstained Standard 1 ml LC5677 BenchMark™ Protein Ladder 2 x 250 µl 10747-012 MagicMark™ Western Protein Standard 250 µl LC5600 MultiMark® Multi-Colored Protein Standard 500 µl LC5725 SeeBlue® Plus2 Pre-Stained Protein Standard 500 µl LC5925 BenchMark™ Pre-Stained Protein Ladder 2 x 250 µl 10748-010 SeeBlue® Pre-Stained Protein Standard 500 µl LC5625 IEF Marker 3-10 500 µl 39212-01

site, enabling you to detect them simultaneously with your target protein using the

effective pI determination

same antibody conjugate and protocol.

The IEF Marker 3-10 (figure 1H) allows you

There’s no need for extra reagents or mark-

to determine the isoelectric point (pI) of

ing and aligning steps. In addition, the non-

unknown protein samples. It includes 13

dye modified MagicMark™ bands provide you

isoforms from 9 purified proteins with pIs

with the most accurate molecular weight

ranging from 3 to 10, eliminating the need

* The BenchMark™ Pre-Stained Protein Ladder is designed for use in the Tris-Glycine gel system. Lot-specific molecular weight values are provided in the product insert. † Apparent molecular weight values vary depending upon electrophoresis gel/buffer system. These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or web site, www.invitrogen.com). By the use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses.

www.invitrogen.com

Expressions 10.3

new product

18

Platinum® PCR SuperMix 96

High-throughput PCR made simple

P

latinum® PCR SuperMix 96 is a complete hot-start PCR reaction mix aliquoted into 96-well plates designed to save you time. It offers the high yield and specificity of Platinum® Taq

DNA Polymerase with the convenience of a PCR master mix in a flexible, high-throughput format. easy to use

figure 1 - superior yield with Platinum® PCR SuperMix 96 vs. other 96-well hot-start master mix

Platinum® PCR SuperMix 96 contains recombinant Taq DNA Polymerase, Platinum® Taq

ABgene Pre-Aliquoted ThermoStart® PCR Master Mix

antibodies, dNTPs, magnesium, and buffers, all pre-mixed and pre-aliquoted into 96-well

bp:

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353

Invitrogen Platinum® PCR SuperMix 96

532 1026 1600 2700 3600

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353

532 1026 1600 2700 3600

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Targets:

plates (22.5 µl/per well). This eliminates the many tedious set-up steps necessary for highthroughput PCR. These plates are available

βactin–353 bp GAPDH–532 bp βactin–1026 bp CBP–1600 bp THRC–2700 bp HTF4–3600 bp

2 kb 1 kb

either in a skirted or non-skirted (perforated) format. The perforations allow you to split the plates into 24-well increments so you use

Various cDNA targets were amplified according to manufacturer’s instructions from 1 µg total HeLa RNA Marker lanes are 1 kb Plus DNA Ladder (Invitrogen).

only the number of reactions you need. With figure 2 - high specificity and yield with Platinum® PCR SuperMix 96

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ment. The Platinum® Taq antibody-mediated

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Rhod-4100 bp

Rhod-2579 bp

P53-2380 bp

P53-1587 bp

Pr1.3-264 bp

high-yield, high-throughput PCR experi-

Invitrogen Platinum® PCR SuperMix 96

Rhod-4100 bp

Now you can amplify all your targets in one

Rhod-2579 bp

Pr1.3-264 bp

save time

P53-2380 bp

Amersham Ready-to-go™ PCR beads

Rhod-670 bp

high-throughput PCR applications.

P53-1587 bp

Platinum® reliability and yield for all your

Rhod-670 bp

Platinum® PCR SuperMix 96, you’ll get proven

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hot-start reaction allows room temperature set-up and eliminates the extra pre-incubation steps associated with most hot-start

2 kb 1 kb

enzymes. You’ll save time by eliminating hundreds of pipeting steps in addition to the 10-minute pre-incubation that is required with some other hot-start enzymes.

Comparison of genomic DNA amplification from K562 cells. Reactions (25 µl) were amplified according to manufacturer’s recommendations. The amount of genomic DNA added was 10 ng for