User Manual - Thermo Fisher Scientific
pENTR™5′-TOPO® TA Cloning Kit
Five-minute TOPO® Cloning of Taq polymeraseamplified PCR products into an entry vector for the
ViraPower™ Promoterless Lentiviral Gateway® and
MultiSite Gateway® Systems
Catalog nos. K591-10, K591-20, and K5910-00
Version C
6 June 2007
25-0744
Corporate Headquarters
Invitrogen Corporation
1600 Faraday Avenue
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
Table of Contents ........................................................................................................................................................ ii
Table of Contents ....................................................................................................................................................... iii
TOPO® Cloning Procedure for Experienced Users ................................................................................................ v
Kit Contents and Storage .........................................................................................................................................vii
Accessory Products .................................................................................................................................................... x
Introduction ....................................................................................................................... 1
Overview...................................................................................................................................................................... 1
Experimental Outline ................................................................................................................................................. 5
Methods.............................................................................................................................. 6
Designing PCR Primers ............................................................................................................................................. 6
Producing PCR Products ........................................................................................................................................... 8
Setting Up the TOPO® Cloning Reaction............................................................................................................... 10
Transforming One Shot® Competent E. coli .......................................................................................................... 12
Analyzing Transformants........................................................................................................................................ 15
Guidelines to Perform the MultiSite Gateway® LR Recombination Reaction .................................................. 16
Troubleshooting .............................................................................................................. 17
Appendix .......................................................................................................................... 19
Performing the Control Reactions .......................................................................................................................... 19
Gel Purifying PCR Products.................................................................................................................................... 21
Addition of 3′ A-Overhangs Post-Amplification ................................................................................................. 23
Map and Features of pENTR™5′-TOPO® ............................................................................................................... 24
Recipes........................................................................................................................................................................ 26
Technical Support..................................................................................................................................................... 27
Purchaser Notification ............................................................................................................................................. 28
Gateway® Clone Distribution Policy...................................................................................................................... 30
References .................................................................................................................................................................. 31
iii
iv
TOPO® Cloning Procedure for Experienced Users
Introduction
This quick reference sheet is provided for experienced users of the TOPO® Cloning
procedure. If you are performing the TOPO® Cloning procedure for the first time, we
recommend that you follow the detailed protocols provided in the manual.
Step
Action
Produce PCR product
Amplify DNA encoding a eukaryotic promoter of choice using Taq polymerase and
your own protocol. End the PCR reaction with a final 7 to 10 minute extension step.
Perform the TOPO®
Cloning Reaction
1.
Set up one of the following TOPO® Cloning reactions using the reagents in the
order shown. For electroporation, dilute Salt Solution 4-fold to prepare Dilute
Salt Solution.
Reagent
0.5 to 4 µl
0.5 to 4 µl
Salt Solution
1 µl
--
Dilute Salt Solution
--
1 µl
Sterile Water
to a final volume of 5 µl
to a final volume of 5 µl
pENTR 5′-TOPO Vector
1 µl
1 µl
Total volume
6 µl
6 µl
®
2.
3.
Mix gently and incubate for 5 minutes at room temperature.
Place on ice and proceed to transform One Shot® TOP10 chemically competent
E. coli, below.
1.
2.
For each transformation, thaw one vial of One Shot® TOP10 E. coli cells on ice.
Add 2 µl of the TOPO® Cloning reaction into a vial of One Shot® TOP10
chemically competent E. coli and mix gently.
Incubate on ice for 5 to 30 minutes.
Heat-shock the cells for 30 seconds at 42°C without shaking. Immediately
transfer the tube to ice.
Add 250 µl of room temperature S.O.C. Medium.
Incubate at 37°C for 1 hour with shaking.
Spread 10-50 µl of bacterial culture on a prewarmed LB agar plate containing
50 µg/ml kanamycin, and incubate overnight at 37°C.
3.
4.
5.
6.
7.
Control Reaction
For Electroporation
Fresh PCR product
™
Transform One Shot®
TOP10 Chemically
Competent E. coli
For Chemical
Transformation
We recommend using the Control PCR Template and the Control PCR Primers
included with the kit to perform the control reaction. See the protocol on pages 1920 for instructions.
v
vi
Kit Contents and Storage
Types of Kits
This manual is supplied with the following kits.
Kit
™
®
Catalog No.
®
K591-20
pENTR 5’-TOPO TA Cloning Kit
™
®
ViraPower Promoterless Lentiviral Gateway Vector Kit
K591-10
ViraPower™ Promoterless Lentiviral Gateway® Expression Kit
K5910-00
Note: Catalog nos. K591-10 and K5910-00 also include ViraPower™ Promoterless Lentiviral
components required for production of a lentiviral expression construct. For more
information about the ViraPower™ Promoterless Lentiviral components, refer to the
ViraPower™ Promoterless Lentiviral Gateway® Expression System manual. This manual is
supplied with the ViraPower™ Promoterless Lentiviral Gateway® Kits, but is also available for
downloading from our Web site (www.invitrogen.com) or by contacting Technical Support
(see page 27).
Shipping/Storage
Each pENTR™5′-TOPO® TA Cloning® Kit is shipped on dry ice, and contains two
boxes as described below. Upon receipt, store the boxes as detailed below.
Box
1
2
Item
™
®
Storage
®
pENTR 5′-TOPO TA Cloning Reagents
®
One Shot TOP10 Chemically Competent E. coli
-20°C
-80°C
continued on next page
vii
Kit Contents and Storage, continued
pENTR™5′-TOPO®
Reagents
The following reagents are supplied with the pENTR™5′-TOPO® vector (Box 1). Note
that the user must supply Taq polymerase. Store at -20°C.
Item
Primer Sequences
Concentration
Amount
pENTR 5′-TOPO vector,
TOPO®-adapted
5-10 ng/µl linearized plasmid DNA in:
50% glycerol
50 mM Tris-HCl, pH 7.4 (at 25°C)
1 mM EDTA
1 mM DTT
0.1% Triton X-100
100 µg/ml BSA
30 µM phenol red
20 µl
10X PCR Buffer
100 mM Tris-HCl, pH 8.3 (at 42°C)
500 mM KCl
25 mM MgCl2
0.01% gelatin
100 µl
dNTP Mix
12.5 mM dATP
12.5 mM dCTP
12.5 mM dGTP
12.5 mM dTTP
neutralized at pH 8.0 in water
10 µl
Salt Solution
1.2 M NaCl
0.06 M MgCl2
50 µl
Sterile Water
---
1 ml
GW1 Primer
0.1 µg/µl in TE Buffer, pH 8.0
20 µl
GW3 Primer
0.1 µg/µl in TE Buffer, pH 8.0
20 µl
Control PCR Primers
0.1 µg/µl each in TE Buffer, pH 8.0
10 µl
Control PCR Template
0.05 µg/µl in TE Buffer, pH 8.0
10 µl
™
®
The table below provides the sequences of GW1 and GW3 primers.
Primer
Sequence
pMoles Supplied
GW1
5′-GTTGCAACAAATTGATGAGCAATGC-3′
260
GW3
5′-TTAATATATTGATATTTATATCATTTTACG-3′
219
continued on next page
viii
Kit Contents and Storage, continued
One Shot® TOP10
Reagents
The following reagents are included with the One Shot® TOP10 Chemically
Competent E. coli kit. Transformation efficiency is ≥ 1 x 109 cfu/µg plasmid DNA.
Store at -80°C.
Reagent
Genotype of
TOP10 Strain
Composition
Amount
S.O.C. Medium
(may be stored at room
temperature or +4°C)
2% Tryptone
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM glucose
6 ml
TOP10 cells
--
21 x 50 µl
pUC19 Control DNA
10 pg/µl in 5 mM Tris-HCl,
0.5 mM EDTA, pH 8
50 µl
F- mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacΧ74 recA1 araD139 ∆(ara-leu)7697
galU galK rpsL (StrR) endA1 nupG
ix
Accessory Products
Introduction
The products listed in this section may be used with the pENTR™5′-TOPO® TA
Cloning® Kit. For more information, refer to our Web site (www.invitrogen.com) or
contact Technical Support (see page 27).
Additional
Products
Some of the reagents supplied in the pENTR™5′-TOPO® TA Cloning® Kit and other
reagents suitable for use with the kit are available separately from Invitrogen.
Ordering information for these reagents is provided below.
Note: Other reagent quantities may be available.
Item
®
Platinum Taq DNA Polymerase
Catalog no.
100 reactions
10966-018
250 reactions
10966-026
500 reactions
10966-034
100 units
10342-053
250 units
10342-012
500 units
10342-020
100 units
11304-011
500 units
11304-029
10 reactions
C4040-10
20 reactions
C4040-03
One Shot TOP10 Electrocompetent E. coli
10 reactions
C4040-50
Kanamycin Sulfate
100 ml (10 mg/ml)
15160-054
100 reactions
K2100-01
20 reactions
12538-020
100 reactions
12538-100
1 kit
12537-023
Taq DNA Polymerase, Recombinant
Platinum® Taq DNA Polymerase High Fidelity
One Shot® TOP10 Chemically Competent E. coli
®
™
PureLink HQ Mini Plasmid Purification Kit
®
™
Gateway LR Clonase II Plus Enzyme Mix
®
MultiSite Gateway Three-Fragment Vector
Construction Kit
x
Quantity
Introduction
Overview
Introduction
The pENTR™5’-TOPO® TA Cloning® Kit combines Invitrogen’s TOPO® Cloning
and MultiSite Gateway® Technologies to facilitate five-minute, one-step cloning of
a Taq polymerase-amplified PCR product encoding a eukaryotic promoter of
interest into a MultiSite Gateway® entry vector with ≥ 85% efficiency. Once cloned,
your eukaryotic promoter may be transferred from the pENTR™5′-TOPO® vector to
a suitable MultiSite Gateway® destination vector in a MultiSite Gateway® LR
recombination reaction with other suitable entry clone(s) to generate an expression
construct.
For more information about how TOPO® Cloning works and the MultiSite
Gateway® Technology, see the rest of this section.
The MultiSite
Gateway®
Technology
The Gateway® Technology is a universal cloning system that takes advantage of
the site-specific recombination properties of bacteriophage lambda (Landy, 1989)
to provide a rapid and highly efficient way to move a single DNA sequence of
interest into multiple vector systems. The MultiSite Gateway® Technology uses
modifications of the Gateway® Technology to allow simultaneous cloning of
multiple DNA fragments in a defined order and orientation to facilitate creation
of an expression construct that expresses a gene of interest from your promoter of
choice. To express a gene of interest using the MultiSite Gateway® Technology,
you will:
1.
TOPO® Clone a Taq-amplified PCR product encoding a eukaryotic promoter
of choice into the pENTR™5′-TOPO® vector to generate a 5′ entry clone.
2.
Generate an entry clone containing your gene of interest using one of the
standard Gateway® entry vectors available from Invitrogen.
Note: If you are using the MultiSite Gateway® Three-Fragment Vector Construction
Kit, also generate an entry clone containing a 3′ element of interest (e.g.
polyadenylation signal) using the reagents supplied in the kit.
3.
Perform a MultiSite Gateway® LR recombination reaction between your 5′
entry clone, other suitable entry clone(s), and the appropriate MultiSite
Gateway® destination vector to generate an expression construct.
4.
Introduce your expression construct into mammalian cells and express your
recombinant protein.
For more information about the MultiSite Gateway® Technology, refer to the
ViraPower™ Promoterless Lentiviral Gateway® Expression System or MultiSite
Gateway® Three-Fragment Vector Construction Kit manuals, as appropriate. Both
manuals are available for downloading from www.invitrogen.com or by
contacting Technical Support (see page 27).
continued on next page
1
Overview, continued
Features of the
pENTR™5′-TOPO®
Vector
att Sites in
pENTR™5′-TOPO®
Features of the pENTR™5′-TOPO® vector include:
•
TOPO® Cloning site for rapid and efficient cloning of a Taq-amplified PCR
product encoding a promoter of choice
•
attL4 and attR1 sites to allow two-fragment or three-fragment recombination
with appropriate entry clone(s) and a MultiSite Gateway® destination vector
to generate an expression construct
•
Primer binding sites within the attL4 and attR1 sites for sequencing using the
GW1 and GW3 primers
•
rrnB transcription termination sequences to prevent basal expression of the
PCR product of interest in E. coli
•
Kanamycin resistance gene for selection in E. coli
•
pUC origin for high-copy replication of the plasmid in E. coli
In the Gateway® Technology, recombinational cloning is mediated via optimized
att sites. To accommodate simultaneous recombinational cloning of multiple DNA
fragments in the MultiSite Gateway® Technology, these att sites have been further
modified and optimized. Modifications include alterations to both the sequence
and length of the att sites, resulting in the creation of “new” att sites exhibiting
enhanced specificities and the improved efficiency required to permit cloning of
multiple DNA fragments in a single reaction.
While traditional Gateway® entry vectors contain attL1 and attL2 sites and may be
recombined with any standard Gateway® destination vector to generate an
expression construct, the pENTR™5′-TOPO® vector contains attL4 and attR1 sites
(refer to the diagram on page 7 for the sequence of the att sites). Because of the
different att sites, entry clones generated in pENTR™5′-TOPO® are only
suitable for use in MultiSite Gateway® applications (see the next page) and not
in other standard Gateway® applications.
continued on next page
2
Overview, continued
Applications for
pENTR™5′-TOPO®
Entry Clones
Entry clones generated in the pENTR™5′-TOPO® vector are suitable for use in the
following MultiSite Gateway® applications. For an illustration, see the figure
below.
•
Combine a pENTR™5′-TOPO® entry clone containing a promoter of interest
with an attL1 and attL2-flanked entry clone containing a gene of interest and
pLenti6/R4R2/V5-DEST to create a lentiviral expression construct. Use the
lentiviral construct to facilitate expression of your recombinant protein in
dividing or non-dividing mammalian cells. For more information, refer to the
ViraPower™ Promoterless Lentiviral Gateway® Expression System manual.
•
Combine a pENTR™5′-TOPO® entry clone containing a promoter of interest
with an attL1 and attL2-flanked entry clone containing a gene of interest, an
attR2 and attL3-flanked entry clone containing a 3′ element of interest, and
pDEST™R4-R3 to create an expression construct. Use the expression construct
to facilitate expression of your recombinant protein in mammalian cells. For
more information, refer to the MultiSite Gateway® Three-Fragment Vector
Construction Kit manual.
promoter
T
T
attR1
attL4
®
pENTR -5’-TOPO
TM
ori
kan
ViraPowerTM Promoterless
Lentiviral Gateway®
Expression System
ori
promoter
kan
ori
kan
ori
pENTR -gene
pENTRTM-5’-promoter
attL4
MultiSite Gateway®
Three-Fragment Vector
Construction Kit
attR1
gene
attL1
attL2
attL4
promoter
kan
ori
kan
pENTRTM-3’ element
pENTRTM-5’-promoter
TM
attR1
gene
attR2
gene
attL1
attL3
attL2
pENTRTM-gene
ccdB
attR4
attR2
pLenti/R4R2/V5-DEST
ori
kan
ori
CmR
ccdB
attR4
amp
CmR
amp
ori
LR ClonaseTM II Plus
attR3
TM
pDEST R4-R3
LR ClonaseTM II Plus
attB4
promoter
attB1
gene
Your expression clone
ori
attB2
attB4
promoter
amp
attB1
gene
attB2 3’ element attB3
Your expression clone
ori
amp
3
Overview, continued
How
Topoisomerase I
Works
The pENTR™5′-TOPO® vector is supplied linearized with:
•
Single 3′-thymidine (T) overhangs for TOPO® TA Cloning®
•
Topoisomerase I covalently bound to the vector (referred to as “activated”
vector)
Taq polymerase has a non-template-dependent terminal transferase activity that
adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized
vector supplied in this kit has single, overhanging 3′ deoxythymidine (T) residues.
This allows PCR inserts to ligate efficiently with the vector.
Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites
(CCCTT) and cleaves the phosphodiester backbone in one strand (Shuman, 1991).
The energy from the broken phosphodiester backbone is conserved by formation of
a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl
residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA
and enzyme can subsequently be attacked by the 5′ hydroxyl of the original
cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994).
TOPO® Cloning exploits this reaction to efficiently clone PCR products.
Topoisomerase
Tyr-274
P
CCCTT
GGGA A
O
OH
PCR Product
HO
Tyr-274
Topoisomerase
4
O
A AGGG
TTCCC
P
Experimental Outline
Flow Chart
The flow chart below describes the general steps required to produce and TOPO®
Clone your Taq-amplified PCR product. Remember that the PCR product should
encode a eukaryotic promoter of interest.
Produce your PCR product
TOPO® Cloning Reaction:
Mix together PCR product and pENTRTM5’-TOPO® vector
Incubate 5 minutes
at room temperature
Transform into competent E. coli cells
Select and analyze colonies
Choose a positive transformant and
isolate plasmid DNA
Proceed to the MultiSite Gateway® LR
recombination reaction
5
Methods
Designing PCR Primers
Introduction
Before you use the pENTR™5′-TOPO® TA Cloning® Kit, you must first design PCR
primers and produce your PCR product. Guidelines are provided in this section to
help you design PCR primers.
Factors to
Consider
It is important to properly design your PCR primers to ensure that you obtain the
PCR product you need for your studies. Consider the following when designing
your PCR primers:
•
Remember that the pENTR™5′-TOPO® entry clone will be flanked by attL4
and attR1 sites, and can only be used in a MultiSite Gateway® LR
recombination reaction with one or more types of entry clones and a suitable
destination vector.
MEND
ION
AT
RECOM
Example: The pENTR™5′-TOPO® entry clone may be used in a MultiSite Gateway® LR
recombination reaction with an attL1 and attL2-flanked entry clone containing the
gene of interest and the pLenti6/R4R2/V5-DEST vector.
Important
•
Your PCR product should encode a eukaryotic promoter of interest suitable
for controlling expression of the downstream gene of interest. Make sure that
the PCR product contains all sequences (e.g. TATA box, transcription factor
binding sites, etc.) necessary to regulate the downstream gene of interest
(after MultiSite Gateway® LR recombination).
•
Your PCR product should contain a transcriptional start site (if one is not
included in the entry clone containing your gene of interest).
•
Your PCR product should not contain an ATG for translation initiation (if one
is included in the entry clone containing your gene of interest).
If you are planning to use your pENTR™5′-TOPO® entry clone in a MultiSite
Gateway® LR reaction with an attL1 and attL2-flanked entry clone and the
pLenti6/R4R2/V5-DEST vector to generate a lentiviral expression vector (i.e.
ViraPower™ Promoterless Lentiviral Gateway® Kits), note that the size of your
insert DNA (i.e. promoter + gene of interest) can influence the viral titer. To
obtain efficient lentiviral packaging, make sure that the combined size of your
promoter + gene of interest does not exceed 4.5-5 kb. Larger inserts may not be
properly packaged and may result in lower lentiviral titers.
When synthesizing PCR primers, do not add 5´ phosphates to the primers as this
will prevent the synthesized PCR product from ligating into the pENTR™5’TOPO® vector.
continued on next page
6
Designing PCR Primers, continued
TOPO® Cloning
Site for
pENTR™5′-TOPO®
Use the diagram below to help you design PCR primers and produce your PCR
product for TOPO® Cloning into the pENTR™5’-TOPO® vector. Note that the
pENTR™5′-TOPO® vector is supplied linearized between nucleotides 705 and 706.
Features of the TOPO® Cloning Region:
•
Restriction sites are labeled to indicate the actual cleavage site.
•
The binding sites for the GW1 and GW3 primers included in the kit are
labeled.
Note: When propagating the pENTR™5′-TOPO® vector and entry clones, we have found
that one nucleotide within the GW1 priming site can vary in sequence between G and A
(highlighted in bold below). This single nucleotide change does not affect the efficiency
of MultiSite Gateway® LR recombination or the functionality of the GW1 sequencing
primer.
•
The shaded region corresponds to the DNA sequences that will be transferred
from the clone into the MultiSite Gateway® destination vector after MultiSite
Gateway® LR recombination.
For a map and a description of the features of pENTR™5’-TOPO®, see page 24. The
sequence of pENTR™5′-TOPO® is available for downloading from our Web site
(www.invitrogen.com) or by contacting Technical Support (see page 27).
M13 forward (-20) priming site
501
TAACGCTAGC ATGGATGTTT TCCCAGTCAC GACGTTGTAA AACGACGGCC AGTCTTAAGC TCGGGCCCGA GTTAACGCTA
GW1 priming site
attL4
581
CCATGGAGCT CCAAATAATG ATTTTATTTT GACTGATAGT GACCTGTTCG TTGCAACAAA TTGATAAGCA ATGCTTTTTT
EcoR I
661
EcoR I
ATAATGCCAA CTTTG TAT AGA AAA GTT GGC TCC GAA TTC GCC CTT
TTT CAA CCG AGG CTT AAG CGG GA A
Lys Val Gly Ser Glu Phe Ala Leu
attR1
PCR product
A AG GGC GAA TTC
TTC CCG CTT AAG
Lys Gly Glu Phe
GW3 priming site
718
GAC CCA AGT TTG TAC AAAAAAGT TGAACGAGAA ACGTAAAATG ATATAAATAT CAATATATTA AATTAGATTT
CTG GGT TCA AAC ATG TTTTTTCA
Asp Pro Ser
791
TGCATAAAAA ACAGACTACA TAATACTGTA AAACACAACA TATGCAGTCA CTATGAACCA ACTACTTAGA TGGTATTAGT
871
GACCTGTAGA ATTAATTCGA GCTCTAGAGC TGCAGGGCGG CCGCGATATC CCCTATAGTG AGTCGTATTA CATGGTCATA
951
GCTGTTTCCT GGCAGCTCTG
M13 reverse priming site
If you have used other Gateway® entry vectors, note that the sequences within
the att sites may vary slightly. The efficiency of LR recombination remains the
same.
7
Producing PCR Products
Introduction
Once you have synthesized appropriate PCR primers, you may use the primers
and a suitable DNA polymerase to produce your PCR product. Remember that
your PCR product must have single 3′ A-overhangs.
Materials Supplied You will need the following reagents and equipment for PCR. Note: dNTPs
(adjusted to pH 8) are provided in the kit.
by the User
•
Taq polymerase or other suitable DNA polymerase
Note: For improved specificity and higher yields, we recommend using Platinum®
Taq DNA Polymerase available from Invitrogen (see page x for ordering information)
to generate your PCR product.
Polymerase
Mixtures
•
Thermocycler
•
DNA template (containing the desired promoter region)
•
PCR primers suitable for amplification of the desired region
You may use a polymerase mixture containing Taq polymerase and a
proofreading polymerase to produce your PCR product; however, the mixture
must contain a ratio of Taq polymerase:proofreading polymerase in excess of 10:1
to ensure the presence of 3′ A-overhangs on the PCR product. We recommend
using Platinum® Taq DNA Polymerase High Fidelity available from Invitrogen
(see page x for ordering information).
If you use polymerase mixtures that do not have enough Taq polymerase or a
proofreading polymerase only, you may add 3′ A-overhangs to your PCR
product using the method on page 23.
Producing PCR
Products
1.
Set up the following 50 µl PCR reaction. Use less DNA if you are using
plasmid DNA as a template and more DNA if you are using genomic DNA as
a template. Use the cycling parameters suitable for your primers and
template. Be sure to include a 7 to 30 minute extension at 72°C after the last
cycle to ensure that all PCR products are full-length and 3′ adenylated.
DNA Template
10-100 ng
10X PCR Buffer
5 µl
0.5 µl
dNTP Mix (50 mM)
PCR primers (100-200 ng each)
Sterile water
add to a final volume of 49 µl
Taq Polymerase (1 U/µl)
Total volume
2.
1 µM each
1 µl
50 µl
Use agarose gel electrophoresis to verify the quality of your PCR product.
You should see a single, discrete band of the correct size. If you do not see a
single band, refer to the Note on the next page.
continued on next page
8
Producing PCR Products, continued
If you do not obtain a single, discrete band from your PCR, try the following:
•
Optimize your PCR to eliminate multiple bands and smearing (Innis et al.,
1990). The PCR Optimizer™ Kit available from Invitrogen (Catalog no. K122001) incorporates many of the recommendations found in this reference. For
more information, refer to our Web site (www.invitrogen.com) or contact
Technical Support (see page 27).
•
Gel-purify your fragment using one of the methods on pages 21-22. Take
special care to avoid sources of nuclease contamination.
9
Setting Up the TOPO® Cloning Reaction
Introduction
Once you have produced the desired PCR product, you are ready to TOPO® Clone
it into the pENTR™5′-TOPO® vector and transform the recombinant vector into
One Shot® TOP10 competent E. coli. You should have everything you need set up
and ready to use to ensure that you obtain the best possible results. We suggest
that you read this section and the section entitled Transforming One Shot®
Competent E. coli (pages 12-13) before beginning. If this is the first time you have
TOPO® Cloned, perform the control reactions on pages 19-20 in parallel with your
samples.
We have found that including salt (200 mM NaCl, 10 mM MgCl2) in the TOPO®
Cloning reaction can increase the number of transformants 2- to 3-fold. In
addition, incubating the reaction mixture for greater than 5 minutes in the
presence of salt can also increase the number of transformants. In contrast, in
experiments performed without salt, the number of transformants decreases as
the incubation time increased beyond 5 minutes.
Including salt in the TOPO® Cloning reaction allows for longer incubation times
because it prevents topoisomerase I from rebinding and potentially nicking the
DNA after ligating the PCR product and dissociating from the DNA. This yields
more intact DNA molecules, leading to higher transformation efficiencies.
Using Salt
Solution in the
TOPO® Cloning
Reaction
You will perform TOPO® Cloning in a reaction buffer containing salt (i.e. using the
stock salt solution provided in the kit). Note that the amount of salt added to the
TOPO® Cloning reaction varies depending on whether you plan to transform
chemically competent cells (provided) or electrocompetent cells (see page x for
ordering information).
•
If you are transforming chemically competent E. coli, use the stock Salt
Solution as supplied and set up the TOPO® Cloning reaction as directed on
the next page.
•
If you are transforming electrocompetent E. coli, the amount of salt in the
TOPO® Cloning reaction must be reduced to 50 mM NaCl, 2.5 mM MgCl2 to
prevent arcing during electroporation. Dilute the stock Salt Solution 4-fold
with water to prepare a 300 mM NaCl, 15 mM MgCl2 Dilute Salt Solution. Use
this Dilute Salt Solution to set up the TOPO® Cloning reaction as directed on
the next page.
continued on next page
10
Setting Up the TOPO® Cloning Reaction, continued
Materials Needed
Performing the
TOPO® Cloning
Reaction
You should have the following materials on hand before beginning:
•
Your PCR product (freshly prepared)
•
The pENTR™5′-TOPO® vector (supplied with the kit, Box 1; keep at -20°C until
use)
•
Salt Solution (supplied with the kit, Box 1) or Dilute Salt Solution (see previous
page), as appropriate
•
Sterile water (supplied with the kit, Box 1)
Use the procedure below to perform the TOPO® Cloning reaction. Set up the
TOPO® Cloning reaction using the reagents in the order shown, and depending on
whether you plan to transform chemically competent E. coli or electrocompetent
E. coli.
Note: The red color of the TOPO® vector solution is normal and is used to visualize the
solution.
Reagent*
Chemically Competent E. coli
Electrocompetent E. coli
Fresh PCR product
0.5 to 4 µl
0.5 to 4 µl
Salt Solution
1 µl
--
Dilute Salt Solution (1:4)
--
1 µl
Sterile Water
add to a final volume of 5 µl
add to a final volume of 5 µl
pENTR™5′-TOPO® vector
1 µl
1 µl
Final volume
6 µl
6 µl
*Store all reagents at –20°C when finished. Salt solution and water can be stored at room temperature or +4°C.
1.
Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C).
Note: For most applications, a 5 minute incubation will yield a sufficient number of
colonies for analysis. Depending on your needs, the length of the TOPO® Cloning
reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR
products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are
TOPO® Cloning a pool of PCR products, increasing the reaction time may yield more
colonies.
2.
Place the reaction on ice and proceed to Transforming One Shot® Competent
E. coli, next page.
Note: You may store the TOPO® Cloning reaction at –20°C overnight.
11
Transforming One Shot® Competent E. coli
Introduction
Once you have performed the TOPO® Cloning reaction (previous page), transform
your pENTR™5′-TOPO® construct into competent E. coli. One Shot® TOP10
Chemically Competent E. coli are included with the kit to facilitate transformation.
You may also transform electrocompetent cells, if desired (see page x for ordering
information). Protocols to transform chemically competent or electrocompetent
E. coli are provided in this section.
Materials Needed
In addition to general microbiological supplies (i.e. plates, spreaders), you will
need the following reagents and equipment:
•
TOPO® Cloning reaction (from Step 2, previous page)
•
One Shot® TOP10 chemically competent E. coli (supplied with the kit, Box 2)
•
S.O.C. Medium (supplied with the kit, Box 2)
•
pUC19 positive control (to check transformation efficiency, if desired;
supplied with the kit, Box 2)
•
42°C water bath (or electroporator with cuvettes, optional)
•
15 ml sterile, snap-cap plastic culture tubes (for electroporation only)
•
LB plates containing 50 µg/ml kanamycin (two for each transformation; see
page 26 for a recipe to prepare LB plates)
•
LB plates containing 100 µg/ml ampicillin (if transforming pUC19 control)
•
37°C shaking and non-shaking incubator
There is no blue-white screening for the presence of inserts. Most transformants
will contain recombinant plasmids with the PCR product of interest cloned into
the vector. The GW1 and GW3 primers are included in the kit to allow you to
sequence across an insert in the TOPO® Cloning site to confirm orientation.
Preparing for
Transformation
For each transformation, you will need one vial of One Shot® competent cells and
two selective plates.
•
Equilibrate a water bath to 42°C (for chemical transformation) or set up your
electroporator if you are using electrocompetent E. coli.
•
Warm the vial of S.O.C. Medium to room temperature.
•
Warm LB plates containing 50 µg/ml kanamycin at 37°C for 30 minutes. If you
are including the pUC19 positive control, prewarm LB plates containing
100 µg/ml ampicillin as well.
•
Thaw on ice one vial of One Shot® cells for each transformation.
continued on next page
12
Transforming One Shot® Competent E. coli, continued
One Shot®
Chemical
Transformation
Protocol
Use the following protocol to transform One Shot® TOP10 chemically competent
E. coli.
1.
Add 2 µl of the TOPO® Cloning reaction from Performing the TOPO®
Cloning Reaction, Step 2, page 11 into a vial of One Shot® Chemically
Competent E. coli and mix gently. Do not mix by pipetting up and down.
Note: If you are transforming the pUC19 control plasmid, use 10 pg (1 µl).
2.
Incubate on ice for 5 to 30 minutes.
Note: Longer incubations on ice seem to have a minimal effect on transformation
efficiency. The length of the incubation is at the user’s discretion.
One Shot®
Electroporation
Protocol
3.
Heat-shock the cells for 30 seconds at 42°C without shaking.
4.
Immediately transfer the tubes to ice.
5.
Add 250 µl of room temperature S.O.C. Medium.
6.
Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for
1 hour.
7.
Spread 10-50 µl from each transformation on a prewarmed selective plate and
incubate overnight at 37°C. To ensure even spreading of small volumes, add
20 µl of S.O.C. Medium. We recommend that you plate two different volumes
to ensure that at least one plate will have well-spaced colonies.
8.
An efficient TOPO® Cloning reaction should produce several hundred
colonies. Pick 10 colonies for analysis (see Analyzing Transformants,
page 15).
Use ONLY electrocompetent cells for electroporation to avoid arcing. Do not
use the One Shot® TOP10 chemically competent cells for electroporation.
1.
Add 2 µl of the TOPO® Cloning reaction from Performing the TOPO®
Cloning Reaction, Step 2, page 11 into a sterile microcentrifuge tube
containing 50 µl of One Shot® TOP10 Electrocompetent E. coli and mix gently.
Do not mix by pipetting up and down. Avoid formation of bubbles.
Transfer the cells to a 0.1 cm cuvette.
2.
Electroporate your samples using your own protocol and your electroporator.
Note: If you have problems with arcing, see the next page.
3.
Immediately add 250 µl of room temperature S.O.C. Medium.
4.
Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) and shake for at
least 1 hour at 37°C to allow expression of the kanamycin resistance gene.
5.
Spread 10-50 µl from each transformation on a prewarmed selective plate and
incubate overnight at 37°C. To ensure even spreading of small volumes, add
20 µl of S.O.C. Medium. We recommend that you plate two different volumes
to ensure that at least one plate will have well-spaced colonies.
6.
An efficient TOPO® Cloning reaction should produce several hundred
colonies. Pick 10 colonies for analysis (see Analyzing Transformants,
page 15).
continued on next page
13
MEND
ION
AT
RECOM
Transforming One Shot® Competent E. coli, continued
To prevent arcing of your samples during electroporation, the volume of cells
should be between 50 and 80 µl (0.1 cm cuvettes) or 100 to 200 µl (0.2 cm
cuvettes).
If you experience arcing during transformation, try one of the following
suggestions:
14
•
Reduce the voltage normally used to charge your electroporator by 10%.
•
Reduce the pulse length by reducing the load resistance to 100 ohms.
•
Ethanol precipitate the TOPO® Cloning reaction and resuspend in water prior
to electroporation.
Analyzing Transformants
Analyzing Positive 1. Pick 10 colonies and culture them overnight in LB or SOB medium containing
50 µg/ml kanamycin.
Clones
2.
Isolate plasmid DNA using your method of choice. If you need ultra-pure
plasmid DNA for automated or manual sequencing, we recommend using
Invitrogen’s PureLink™ HQ Mini Plasmid Purification Kit (Catalog No. K210001).
3.
Analyze the plasmids by restriction analysis or PCR to confirm the presence
and correct orientation of the insert.
Note: pENTR™5′-TOPO® contains EcoRI sites flanking the TOPO® Cloning site. You
may use EcoRI digestion to check for the presence of insert, if desired.
Sequencing
Once you have identified the correct clone(s), you may sequence your construct to
confirm that your promoter is cloned in the correct orientation. Use the GW1 and
GW3 primers included in the kit to sequence your insert. The GW1 and GW3
primer binding sites are located within the attL4 and attR1 sites, respectively, and
therefore minimize the amount of vector-encoded DNA that needs to be read to
less than 55 base pairs. (see the diagram on page 7 for the location of the priming
sites).
Alternatively, the pENTR™5′-TOPO® vector also includes M13 forward and
reverse primer binding sites for sequencing using M13 forward (-20) and M13
reverse primers, if desired. Note that the M13 forward and reverse primer binding
sites are located upstream and downstream of the of the attL4 and attR1 sites,
respectively, requiring that at least 130 base pairs of vector-encoded DNA be read
before reaching the insert DNA.
Reminder: When using the GW1 primer for sequencing, note that one nucleotide (position
646) within the primer binding site of the vector can vary in sequence between G and A.
This variation does not affect the functionality of the GW1 sequencing primer or
sequencing results.
Long-Term
Storage
Once you have identified the correct clone, be sure to purify the colony and make
a glycerol stock for long-term storage. We recommend that you store a stock of
plasmid DNA at -20°C.
1.
Streak the original colony out for single colonies on an LB plate containing
50 µg/ml kanamycin.
2.
Isolate a single colony and inoculate into 1-2 ml of LB containing 50 µg/ml
kanamycin.
3.
Grow until culture reaches stationary phase.
4.
Mix 0.85 ml of culture with 0.15 ml of sterile glycerol. Transfer to a cryovial.
5.
Store at -80°C.
15
Guidelines to Perform the MultiSite Gateway® LR
Recombination Reaction
Introduction
Once you have obtained your pENTR™5′-TOPO® entry clone, you may:
•
Perform a two-fragment MultiSite Gateway® LR recombination reaction using
the pENTR™5′-TOPO® entry clone, an attL1 and attL2-flanked entry clone
containing your gene of interest, and the pLenti6/R4R2/V5-DEST destination
vector to generate a lentiviral expression construct. For more information
about pLenti6/R4R2/V5-DEST, refer to the ViraPower™ Promoterless
Lentiviral Gateway® Expression System manual.
•
Perform a three-fragment MultiSite Gateway® LR recombination reaction
using the pENTR™5′-TOPO® entry clone, an attL1 and attL2-flanked entry
clone containing your gene of interest, an attR2 and attL3-flanked entry clone
containing a 3′ element of interest, and the pDEST™R4-R3 destination vector
to generate an expression construct. For more information about pDEST™R4R3 and how to generate an attR2 and attL3-flanked entry clone, refer to the
MultiSite Gateway® Three Fragment Vector Construction Kit manual.
Important
To perform a two-fragment or three-fragment MultiSite Gateway® LR
recombination reaction, you must use the exact combination of entry clone and
destination vector substrates listed above. Note that Gateway® destination vectors
other than the ones listed above cannot be used.
MEND
For optimal results, we recommend performing the MultiSite Gateway® LR
recombination reaction using:
ION
AT
RECOM
General guidelines to perform the MultiSite Gateway® LR recombination reaction
are provided in this section. For detailed instructions, see the appropriate manual
as recommended above.
•
Supercoiled entry clones
•
Supercoiled destination vector
LR Clonase™ II
Plus Enzyme Mix
Use LR Clonase™ II Plus enzyme mix available from Invitrogen to catalyze the
MultiSite Gateway® LR recombination reaction. Note that the LR Clonase™ enzyme
mix used for standard Gateway® LR recombination reactions cannot be used for
MultiSite Gateway® LR recombination reactions. See page x for ordering
information.
E. coli Host
Once you have performed the MultiSite Gateway® LR recombination reaction, you
will transform the reaction mixture into competent E. coli and select for expression
clones. For pLenti6/R4R2/V5-DEST expression clones, use Stbl3™ E. coli for
transformation. For pDEST™R4-R3 expression clones, use any recA, endA E. coli
strain including TOP10, Mach1™-T1R, DH5α™, or equivalent for transformation. Do
not transform the MultiSite Gateway® LR reaction mixture into E. coli strains that
contain the F′ episome (e.g. TOP10F′). These strains contain the ccdA gene and will
prevent negative selection with the ccdB gene.
16
Troubleshooting
TOPO® Cloning
Reaction and
Transformation
The table below lists some potential problems and possible solutions that may
help you troubleshoot the TOPO® Cloning and transformation reactions. To help
evaluate your results, we recommend that you perform the control reactions (see
pages 19-20) in parallel with your samples.
Problem
Few or no colonies obtained
from sample reaction and
the transformation control
gave colonies
Reason
Incomplete extension during
PCR
Solution
Include a final extension step of 7
to 30 minutes during PCR. Longer
PCR products will need a longer
extension time.
Excess (or overly diluted) PCR Reduce (or concentrate) the
product used in the TOPO®
amount of PCR product.
Cloning reaction
PCR primers contain 5′
phosphates
Do not add 5′ phosphates to your
PCR primers.
Used a proofreading polymerase or a Taq/proofreading
polymerase mixture for PCR
•
•
Large PCR product
•
•
•
PCR reaction contains artifacts •
(i.e. does not run as a single
•
band on an agarose gel)
Use Taq polymerase or another
DNA polymerase that leaves 3′
A-overhangs to produce your
PCR product.
Add 3′ A-overhangs to your
blunt PCR product by
incubating with Taq polymerase (see page 23).
Increase the amount of PCR
product used in the TOPO®
Cloning reaction.
Increase the incubation time of
the TOPO® Cloning reaction
from 5 minutes to 30 minutes.
Gel-purify the PCR product to
remove primer-dimers and
other artifacts.
Optimize your PCR conditions.
Gel-purify your PCR product.
continued on next page
17
Troubleshooting, continued
TOPO® Cloning Reaction and Transformation, continued
Problem
Reason
Solution
Few or no colonies obtained
from sample reaction and
the transformation control
gave colonies, continued
PCR product does not contain •
sufficient 3′ A-overhangs even
though you used Taq
polymerase
•
Increase the final extension
time to ensure that all 3′ ends
are adenylated.
Taq polymerase is most
efficient at adding a nontemplate 3′ A next to a C, and
less efficient at adding a
nontemplate 3′ A next to
another A. You may have to redesign your primers so that
they contain a 5′ G instead of a
5′ T (Brownstein et al., 1996).
Large number of incorrect
inserts cloned
PCR cloning artifacts
•
Gel-purify your PCR product
to remove primer-dimers and
smaller PCR products.
Optimize your PCR conditions.
Include a final extension step
of 7 to 30 minutes during PCR.
Longer PCR products will need
a longer extension time.
•
•
Few or no colonies obtained
from sample reaction and
the transformation control
gave no colonies
18
One Shot® competent E. coli
stored incorrectly
Store One Shot® competent E. coli at
-80°C.
If you are using another E. coli
strain, follow the manufacturer’s
instructions.
Did not perform the 1-hour
grow-out period before
plating the transformation
mixture
After the heat-shock step, add
S.O.C. Medium and incubate the
transformation mixture for 1 hour
at 37°C before plating.
Insufficient amount of E. coli
plated
Increase the amount of E. coli
plated.
Transformants plated on
selective plates containing the
wrong antibiotic
Use the appropriate antibiotic for
selection.
Appendix
Performing the Control Reactions
Introduction
We recommend performing the following control TOPO® Cloning reactions the
first time you use the kit to help you evaluate your results. Performing the control
reactions involves producing a control PCR product containing the lac promoter
and the LacZα fragment using the reagents included in the kit. Successful TOPO®
Cloning of the control PCR product in either direction will yield blue colonies on
LB agar plates containing kanamycin and X-gal.
Before Starting
For each transformation, prepare two LB plates containing 50 µg/ml kanamycin
and X-gal (see page 26 for recipes).
Producing the
Control PCR
Product
Use the procedure below to produce the 500 bp control PCR product using Taq
polymerase.
1.
In a 0.5 ml microcentrifuge tube, set up the following 50 µl PCR:
Reagent
Amount
Control DNA Template (50 ng)
1 µl
10X PCR Buffer
5 µl
dNTP Mix
0.5 µl
Control PCR Primers (0.1 µg/µl each)
1 µl
Sterile water
41.5 µl
Taq polymerase (1 U/µl)
1 µl
Total volume
50 µl
2.
Overlay with 70 µl (1 drop) of mineral oil, if required.
3.
Amplify using the following cycling parameters:
Step
4.
Time
Temperature
Initial Denaturation
2 minutes
94°C
Denaturation
1 minute
94°C
Annealing
1 minute
60°C
Extension
1 minute
72°C
Final Extension
7 minutes
72°C
Cycles
1X
25X
1X
Remove 10 µl from the reaction and analyze by agarose gel electrophoresis. A
discrete 500 bp band should be visible. Proceed to the Control TOPO® Cloning
Reactions, next page.
continued on next page
19
Performing the Control Reactions, continued
Control TOPO®
Cloning Reactions
Using the control PCR product produced on the previous page and the
pENTR™5′-TOPO® vector, set up two 6 µl TOPO® Cloning reactions as described
below.
1. Set up control TOPO® Cloning reactions:
Reagent
"Vector Only"
"Vector + PCR Insert"
Sterile Water
4 µl
3 µl
Salt Solution
1 µl
1 µl
Control PCR Product
--
1 µl
pENTR 5′-TOPO vector
1 µl
1 µl
Total volume
6 µl
6 µl
™
®
2. Incubate at room temperature for 5 minutes and place on ice.
3. Transform 2 µl of each reaction into separate vials of One Shot® competent
cells using the procedure on page 13.
4. Spread 10-50 µl of each transformation mix onto LB plates containing
50 µg/ml kanamycin and X-gal. When plating small volumes, add 20 µl of
S.O.C. Medium to ensure even spreading. Be sure to plate two different
volumes to ensure that at least one plate has well-spaced colonies.
5. Incubate overnight at 37°C.
What You Should
See
The “vector + PCR insert” reaction should be produce hundreds of colonies.
Greater than 85% of these will be blue.
The “vector only” reaction should yield very few colonies (< 15% of the vector +
PCR insert plate) and these should be white.
Transformation
Control
20
pUC19 plasmid is included to check the transformation efficiency of the One
Shot® TOP10 competent cells. Transform one vial of One Shot® TOP10 cells with
10 pg of pUC19 using the protocol on page 13. Plate 10 µl of the transformation
mixture plus 20 µl of S.O.C. Medium on LB plates containing 100 µg/ml
ampicillin. Transformation efficiency should be ≥ 1 x 109 cfu/µg DNA.
Gel Purifying PCR Products
Introduction
Smearing, multiple banding, primer-dimer artifacts, or large PCR products
(> 3 kb) may necessitate gel purification. If you wish to purify your PCR product,
be extremely careful to remove all sources of nuclease contamination. There are
many protocols to isolate DNA fragments or remove oligonucleotides. Refer to
Current Protocols in Molecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most
common protocols. Three simple protocols are provided below.
The cloning efficiency may decrease with purification of the PCR product (e.g.
PCR product too dilute). You may wish to optimize your PCR to produce a
single band (see Producing PCR Products, page 8).
Using the
S.N.A.P.™ Gel
Purification Kit
The S.N.A.P.™ Gel Purification Kit available from Invitrogen (Catalog No. K199925) allows you to rapidly purify PCR products from regular agarose gels.
1.
Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel.
Note: Do not use TBE to prepare agarose gels. Borate interferes with the sodium
iodide step, below.
Quick S.N.A.P.™
Method
2.
Cut out the gel slice containing the PCR product and melt it at 65°C in
2 volumes of the 6 M sodium iodide solution.
3.
Add 1.5 volumes Binding Buffer.
4.
Load solution (no more than 1 ml at a time) from Step 3 onto a S.N.A.P.™
column. Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the
supernatant.
5.
If you have solution remaining from Step 3, repeat Step 4.
6.
Add 900 µl of the Final Wash Buffer.
7.
Centrifuge 1 minute at full speed in a microcentrifuge and discard the flowthrough.
8.
Repeat Step 7.
9.
Elute the purified PCR product in 40 µl of TE or sterile water. Use 4 µl for the
TOPO® Cloning reaction and proceed as described on page 11.
An even easier method is to simply cut out the gel slice containing your PCR
product, place it on top of the S.N.A.P.™ column bed, and centrifuge at full speed
for 10 seconds. Use 1-2 µl of the flow-through in the TOPO® Cloning reaction (see
page 11). Be sure to make the gel slice as small as possible for best results.
continued on next page
21
Gel Purifying PCR Products, continued
Low-Melt Agarose
Method
If you prefer to use low-melt agarose, use the procedure below. Note that gel
purification will result in a dilution of your PCR product and a potential loss of
cloning efficiency.
1.
Electrophorese as much as possible of your PCR reaction on a low-melt
agarose gel (0.8 to 1.2%) in TAE buffer.
2.
Visualize the band of interest and excise the band.
3.
Place the gel slice in a microcentrifuge tube and incubate the tube at 65°C
until the gel slice melts.
4.
Place the tube at 37°C to keep the agarose melted.
5.
Add 4 µl of the melted agarose containing your PCR product to the TOPO®
Cloning reaction as described on page 11.
6.
Incubate the TOPO® Cloning reaction at 37°C for 5 to 10 minutes. This is to
keep the agarose melted.
7.
Transform 2 to 4 µl directly into One Shot® competent cells using the method
on page 13.
The cloning efficiency may decrease with purification of the PCR product. You
may wish to optimize your PCR to produce a single band.
22
Addition of 3′ A-Overhangs Post-Amplification
Introduction
Direct cloning of DNA amplified by proofreading polymerases into TOPO® TA
Cloning® vectors is often difficult because proofreading polymerases remove the
3′ A-overhangs necessary for TA Cloning®. Invitrogen has developed a simple
method to clone these blunt-ended fragments.
Before Starting
You will need the following items:
Procedure
•
Taq polymerase
•
A heat block equilibrated to 72°C
•
Phenol-chloroform (optional)
•
3 M sodium acetate (optional)
•
100% ethanol (optional)
•
80% ethanol (optional)
•
TE buffer (optional)
This is just one method for adding 3′ adenines. Other protocols may be suitable.
1.
After amplification with a proofreading polymerase, place vials on ice and
add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to
change the buffer. A sufficient number of PCR products will retain the
3′ A-overhangs.
2.
Incubate at 72°C for 8-10 minutes (do not cycle).
3.
Place on ice and use immediately in the TOPO® Cloning reaction.
Note: If you plan to store your sample overnight before proceeding with TOPO® Cloning,
extract your sample with an equal volume of phenol-chloroform to remove the
polymerases. Ethanol-precipitate the DNA and resuspend in TE buffer using the starting
volume of the PCR.
You may also gel-purify your PCR product after amplification with a
proofreading polymerase. After purification, add Taq polymerase buffer, dATP,
and 0.5 unit of Taq polymerase. Incubate the reaction for 10-15 minutes at 72°C
and use in the TOPO® Cloning reaction.
23
Map and Features of pENTR™5′-TOPO®
pENTR™5′-TOPO®
Map
The figure below shows the features of the pENTR™5′-TOPO® vector. Note that
the pENTR™5′-TOPO® vector is supplied linearized between nucleotides 705 and
706. The complete sequence of pENTR™5′-TOPO® is available for
downloading from www.invitrogen.com or by contacting Technical Support
(see page 27).
CCCTT
GGGA
AGGG
TTCCC
TOPO
attL4
att
R
1
T1
EcoR I
EcoR I
TOPO
pUC ori
T
2
Comments for pENTRTM5’-TOPO®
2680 nucleotides
rrnB T2 transcription terminator: bases 268-295 (c)
rrnB T1 transcription terminator: bases 427-470 (c)
M13 forward (-20) priming site: bases 537-552
attL4: bases 592-691
GW1 priming site: bases 630-654
TOPO® recognition site 1: bases 701-705
TOPO® recognition site 2: bases 706-710
attR1: bases 721-845
GW3 priming site: bases 752-781
M13 reverse priming site: bases 945-961
Kanamycin resistance gene: 1074-1883
pUC origin: bases 2004-2677
pENTR 5’-TOPO®
2680 bp
TM
Kan
c
a my
in
(c) = complementary strand
continued on next page
24
Map and Features of pENTR™5′-TOPO®, continued
Features of
pENTR™5′-TOPO®
pENTR™5′-TOPO® (2680 bp) contains the following elements. All features have
been functionally tested and the vector fully sequenced.
Feature
Benefit
rrnB T1 and T2 transcription termination Reduces potential toxicity in E. coli by
sequences
preventing basal expression of the PCR product.
M13 forward (-20) priming site
Allows sequencing of the insert.
GW1 priming site
Allows sequencing of the insert.
attL4 and attR1 sites
Bacteriophage λ-derived recombination
sequences that have been optimized to allow
recombinational cloning of a DNA fragment in
the entry construct with a suitable MultiSite
Gateway® destination vector in conjunction with
any attL1 and attL2-flanked entry clone (Landy,
1989).
TOPO® Cloning site
Allows rapid cloning of your Taq-amplified PCR
product.
GW3 priming site
Allows sequencing of the insert.
M13 reverse priming site
Allows sequencing of the insert.
Kanamycin resistance gene
Allows selection of the plasmid in E. coli.
pUC origin of replication (ori)
Allows high-copy replication and maintenance
in E. coli.
25
Recipes
LB (Luria-Bertani)
Medium and
Plates
Composition:
1.0% Tryptone
0.5% Yeast Extract
1.0% NaCl
pH 7.0
1.
For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 ml
deionized water.
2.
Adjust the pH of the solution to 7.0 with NaOH and bring the volume up to
1 liter.
3.
Autoclave on liquid cycle for 20 minutes. Allow solution to cool to ~55°C
and add antibiotic, if needed.
4.
Store at room temperature or at +4°C.
LB agar plates
X-Gal Stock
Solution
26
1.
Prepare LB medium as above, but add 15 g/L agar before autoclaving.
2.
Autoclave on liquid cycle for 20 minutes.
3.
After autoclaving, cool to ~55°C, add antibiotic and pour into 10 cm plates.
4.
Let harden, then invert and store at +4°C, in the dark.
5.
To add X-gal to the plate, warm the plate to 37°C. Pipette 40 µl of the
40 mg/ml X-gal stock solution (see below), spread evenly, and let dry for
15 minutes. Protect plates from light.
1.
Dissolve 400 mg of X-gal in 10 ml dimethylformamide to prepare a
40 mg/ml stock solution.
2.
Store at -20°C, protected from light.
Technical Support
Web Resources
Visit the Invitrogen Web site at www.invitrogen.com for:
• Technical resources, including manuals, vector maps and sequences,
application notes, MSDSs, FAQs, formulations, citations, handbooks, etc.
• Complete technical support contact information
• Access to the Invitrogen Online Catalog
• Additional product information and special offers
Contact Us
For more information or technical assistance, call, write, fax, or email. Additional
international offices are listed on our website (www.invitrogen.com).
Corporate Headquarters:
Invitrogen Corporation
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Tel: 1 760 603 7200
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Tel: +44 (0) 141 814 6100
Tech Fax: +44 (0) 141 814 6117
E-mail: [email protected]
MSDS
MSDSs (Material Safety Data Sheets) are available on our Web site at
www.invitrogen.com/msds.
Certificate of
Analysis
Product qualification is described in the Certificate of Analysis (CofA), available
on our Web site by product lot number at www.invitrogen.com/cofa.
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.
27
Purchaser Notification
Introduction
Use of the pENTR™5’-TOPO® TA Cloning® Kit is covered under the licenses detailed
below.
Limited Use Label
License No. 5:
Invitrogen
Technology
The purchase of this product conveys to the buyer the non-transferable 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. Invitrogen
Corporation will not assert a claim against the buyer of infringement of patents owned or
controlled by Invitrogen 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, Invitrogen is willing to accept return of the product with a full
refund. For information on purchasing a license to this product for purposes other than
research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue,
Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500. Email:
[email protected]
28
Purchaser Notification, continued
Limited Use Label
License No. 19:
Gateway® Cloning
Products
This product and its use is the subject of one or more of U.S. Patent Nos. 5,888,732,
6,143,557, 6,171,861, 6,270,969, and 6,277,608 and/or other pending U.S. and foreign
patent applications owned by Invitrogen Corporation. The purchase of this product
conveys to the buyer the non-transferable 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 purchase of this product does not convey a
license under any method claims in the foregoing patents or patent applications, or to use
this product with any recombination sites other than those purchased from Invitrogen
Corporation or its authorized distributor. The right to use methods claimed in the
foregoing patents or patent applications with this product for research purposes only can
only be acquired by the use of ClonaseTM purchased from Invitrogen Corporation or its
authorized distributors. The buyer cannot modify the recombination sequence(s)
contained in this product for any purpose. The buyer cannot sell or otherwise transfer (a)
this product, (b) its components, or (c) materials made by the employment of this product
or its components to a third party or otherwise use this product or its components or
materials made by the employment of this product or its components for Commercial
Purposes. The buyer may transfer information or materials made through the
employment 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.
Notwithstanding the preceding, any buyer who is employed in an academic or
government institution may transfer materials made with this product to a third party
who has a license from Invitrogen under the patents identified above to distribute such
materials. Transfer of such materials and/or information to collaborators does not convey
rights to practice any methods claimed in the foregoing patents or patent applications.
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. Invitrogen Corporation will not assert a claim against the
buyer of infringement of the above patents 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 none of
(i) this product, (ii) any of its components, or (iii) a method claim of the foregoing patents,
was used in the manufacture of such product. Invitrogen Corporation will not assert a
claim against the buyer of infringement of the above patents based upon the use of this
product to manufacture a protein for sale, provided that no method claim in the above
patents was used in the manufacture of such protein. If the purchaser is not willing to
accept the limitations of this limited use statement, Invitrogen is willing to accept return
of the product with a full refund. For information on purchasing a license to use this
product for purposes other than those permitted above, contact Licensing Department,
Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760)
603-7200.
For additional information about Invitrogen’s policy for the use and distribution of
Gateway® Clone
®
®
Distribution Policy Gateway clones, see the section entitled Gateway Clone Distribution Policy, next page.
29
Gateway® Clone Distribution Policy
Introduction
The information supplied in this section is intended to provide clarity
concerning Invitrogen’s policy for the use and distribution of cloned nucleic
acid fragments, including open reading frames, created using Invitrogen’s
commercially available Gateway® Technology.
Gateway® Entry
Clones
Invitrogen understands that Gateway® entry clones, containing attL1 and attL2
sites, may be generated by academic and government researchers for the purpose
of scientific research. Invitrogen agrees that such clones may be distributed for
scientific research by non-profit organizations and by for-profit organizations
without royalty payment to Invitrogen.
Gateway®
Expression
Clones
Invitrogen also understands that Gateway® expression clones, containing attB1
and attB2 sites, may be generated by academic and government researchers for
the purpose of scientific research. Invitrogen agrees that such clones may be
distributed for scientific research by academic and government organizations
without royalty payment to Invitrogen. Organizations other than academia and
government may also distribute such Gateway® expression clones for a nominal
fee ($10 per clone) payable to Invitrogen.
Additional Terms
and Conditions
We would ask that such distributors of Gateway® entry and expression clones
indicate that such clones may be used only for research purposes, that such
clones incorporate the Gateway® Technology, and that the purchase of
Gateway® Clonase™ from Invitrogen is required for carrying out the Gateway®
recombinational cloning reaction. This should allow researchers to readily
identify Gateway® containing clones and facilitate their use of this powerful
technology in their research. Use of Invitrogen’s Gateway® Technology,
including Gateway® clones, for purposes other than scientific research may
require a license and questions concerning such commercial use should be
directed to Invitrogen’s licensing department at 760-603-7200.
30
References
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994)
Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New
York
Brownstein, M. J., Carpten, J. D., and Smith, J. R. (1996) Modulation of Non-Templated Nucleotide
Addition by Taq DNA Polymerase: Primer Modifications that Facilitate Genotyping.
BioTechniques 20, 1004-1010
Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (eds) (1990) PCR Protocols: A Guide to Methods
and Applications, Academic Press, San Diego, CA
Landy, A. (1989) Dynamic, Structural, and Regulatory Aspects of Lambda Site-specific Recombination.
Ann. Rev. Biochem. 58, 913-949
Shuman, S. (1991) Recombination Mediated by Vaccinia Virus DNA Topoisomerase I in Escherichia coli is
Sequence Specific. Proc. Natl. Acad. Sci. USA 88, 10104-10108
Shuman, S. (1994) Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia
DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684
©2004-2007 Invitrogen Corporation. All rights reserved.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
31
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Invitrogen Corporation
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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
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