GeneClip™ U1 Hairpin Cloning Systems InstrucƟ ons for use of Products

Transcription

TECHNICAL MANUAL
GeneClip™ U1
Hairpin Cloning Systems
InstrucƟons for use of Products
C8750, C8760, C8770, C8780 and C8790
Revised 1/14
TM256
tm256.0114:EIVD_TM.qxd
1/28/2014
10:36 AM
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GeneClip™
U1 Hairpin Cloning Systems
All technical literature is available on the Internet at www.promega.com/protocols
Please visit the web site to verify that you are using the most current version of this
Technical Manual. Please contact Promega Technical Services if you have questions on use
of this system. E-mail [email protected].
1. Description..........................................................................................................2
2. Product Components and Storage Conditions ............................................5
3. Oligonucleotide Design ...................................................................................6
A. siRNA Hairpin Target Sequence Selection.......................................................6
B. GeneClip™ Hairpin Oligonucleotide Design ..................................................6
4. Cloning a Hairpin Insert into the pGeneClip™ Vectors...........................8
A. Oligonucleotide Dilution and Annealing .........................................................8
B. Ligation of a Hairpin Insert into the pGeneClip™ Vectors ..........................8
C. Transformation of E. coli with
pGeneClip™ Vectors Containing Inserts...........................................................10
D. Purifying Recombinant Plasmid DNA............................................................12
E. Screening for Inserts Using PstI Digestion.....................................................12
5. Transfection of pGeneClip™ Vector Constructs
in an siRNA Suppression Assay...................................................................13
A. Transient Transfection of the pGeneClip™ Vector Constructs ..................13
B. Stable Transfection of the pGeneClip™
Puromycin, Hygromycin and Neomycin Vector Constructs ......................14
C. Quantitating siRNA Target Gene Suppression .............................................15
6. Troubleshooting...............................................................................................16
7. References .........................................................................................................17
8. Appendix ...........................................................................................................19
A.
B.
C.
D.
E.
F.
G.
H.
pGeneClip™ Vector Maps and Sequence Reference Points ........................19
pGeneClip™ Basic Vector Restriction Enzyme Sites ....................................24
pGeneClip™ Puromycin Vector Restriction Enzyme Sites .........................26
pGeneClip™ Hygromycin Vector Restriction Enzyme Sites.......................28
pGeneClip™ Neomycin Vector Restriction Enzyme Sites...........................30
pGeneClip™ hMGFP Vector Restriction Enzyme Sites ...............................32
Composition of Buffers and Solutions ............................................................34
Related Products.................................................................................................34
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 1/14
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Description
RNA interference (RNAi), a phenomenon in which double-stranded RNA
suppresses expression of a target protein by stimulating specific degradation of
the target mRNA, provides a powerful genetic tool for selectively silencing
gene expression in many eukaryotes (1–6). In mammalian systems, short
interfering RNAs (siRNAs) are the main effectors of the RNAi process (7,8). The
sequence-specific RNAi effect can be observed by introduction of siRNAs into
cells either via transfection or by endogenous expression of 21–23 base
transcripts (8–10).
In vivo expression of siRNAs can be effectively achieved by generating DNA
vectors containing a U1 RNA polymerase promoter, a template for
transcription of an siRNA and a transcription terminator sequence, then
transfecting these into eukaryotic cells. In cells, RNA polymerase II normally
recognizes the U1 promoter to transcribe small nuclear RNAs. The U1 promoter
has been used successfully to generate siRNAs in mammalian cells (11).
GeneClip™ U1 Hairpin Cloning Systems
In the GeneClip™ U1 Hairpin Cloning Systems, siRNAs are expressed as foldback stem-loop structures that are transcribed from the U1 promoter. The
GeneClip™ U1 Hairpin Cloning Systems include linearized plasmids
(pGeneClip™ Vectors) designed for easy and fast cloning of hairpin target
sequences to allow expression of siRNA target sequences in human cells. The
pGeneClip™ Vectors contain the human U1 promoter, which allows
transcription of the hairpin target sequences and generation of hairpin siRNA
in vivo. The GeneClip™ U1 Hairpin Cloning System—Basic is intended for
transient suppression of the gene of interest. The GeneClip™ U1 Hairpin
Cloning System—hMGFP enables easy determination of transfection efficiency
and allows selection of transfected cells by fluorescence-activated cell sorting
(FACS®; 12–14). The other GeneClip™ U1 Hairpin Cloning Systems offer
antibiotic marker options (neomycin, hygromycin or puromycin) for selecting
stably transfected eukaryotic cells so that experimental results do not depend
on transfection efficiency.
Hairpin Insert
Two DNA oligonucleotides, supplied by the user, are synthesized and annealed
to form a DNA insert that contains the hairpin siRNA target sequence. Upon
annealing, the oligonucleotides form ends that are compatible with the ends of
the linearized pGeneClip™ Vector and facilitate a “sticky end” ligation (Figure
1). All of the pGeneClip™ Vectors contain the Ampr gene, which confers
resistance to ampicillin and allows selection in E. coli.
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Screening for Successful Ligation
The GeneClip™ U1 Hairpin Cloning Systems are designed to allow easy
determination of successful ligation. Successful ligation of the annealed
oligonucleotides and the vector results in creation of a second PstI restriction
site in addition to the PstI site already present in the pGeneClip™ Vectors. The
presence of an insert can be confirmed by PstI digestion; clones containing an
insert will produce two bands on an agarose gel.
Features
More Vector Choices: These systems provide vectors containing a variety of
eukaryotic, antibiotic-selectable markers for stable transfection, or hMGFP for
determination of transfection efficiency and selection of transfected cells by
FACS® analysis.
Time Savings: Vectors are supplied predigested to eliminate time-consuming
vector preparation.
Convenience: The system includes T4 DNA Ligase, 2X Rapid Ligation Buffer,
Nuclease-Free Water and Oligo Annealing Buffer in addition to the
pGeneClip™ Vector.
Easier Identification of Desired Clones: A PstI digestion quickly identifies
positive recombinants.
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Annealed Hairpin
Oligonucleotides
5´ TCTC
3´
CT
3´
GACGTC 5´
U1 Promoter
AG
GCA
G
AG
pGeneClip™ Vector
PstI
Ligation
T
AG C
U1 Promoter
TC G
A
New PstI site
CTGC
A
GACG G
TC
Hairpin oligonucleotides
ligated into the
pGeneClip™ Vector
Screening for inserts
by PstI digestion
4714MA
PstI
Figure 1. Overview of the GeneClip™ U1 Hairpin Cloning System protocol.
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Product Components and Storage Conditions
Product
GeneClip™ U1 Hairpin Cloning System—Basic(a,b,c)
Each system contains sufficient reagents for 20 ligation reactions.
•
200µl 2X Rapid Ligation Buffer
• 100 units T4 DNA Ligase
•
1.2µg pGeneClip™ Basic Vector, 50µg/ml
•
1ml Oligo Annealing Buffer
• 1.25ml Nuclease-Free Water
Cat.#
C8750
Product
GeneClip™ U1 Hairpin Cloning System—Puromycin(a,b,c)
•
•
1.2µg pGeneClip™ Puromycin Vector, 50µg/ml
•
Cat.#
C8760
Product
GeneClip™ U1 Hairpin Cloning System—Hygromycin(a,b,c)
•
•
1.2µg pGeneClip™ Hygromycin Vector, 50µg/ml
•
Cat.#
C8770
Product
GeneClip™ U1 Hairpin Cloning System—Neomycin(a,b,c)
•
•
1.2µg pGeneClip™ Neomycin Vector, 50µg/ml
•
Cat.#
C8780
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Product
GeneClip™ U1 Hairpin Cloning System—hMGFP(a,b,d–f)
•
•
1.2µg pGeneClip™ hMGFP Vector, 50µg/ml
•
Cat.#
C8790
Storage Conditions: Store at –20°C.
3.
Oligonucleotide Design
3.A. siRNA Hairpin Target Sequence Selection
Choose a 19–23 nucleotide target sequence that starts with a "G" from the
coding sequence of the gene of interest. General guidelines for target sequence
selection are continuously being developed (15,16).
Although the existing rules for siRNA selection serve as a reliable guide, they do
not ensure that each selected siRNA sequence will reduce gene expression, and
the optimal target sequence may have to be determined experimentally (17).
!
As a negative control for RNA interference, use a nonspecific target sequence or
a scrambled sequence.
3.B. GeneClip™ Hairpin Oligonucleotide Design
The GeneClip™ U1 Hairpin Cloning Systems are designed to allow easy and
fast detection of successful hairpin insertion. Two hairpin oligonucleotides
(oligonucleotides A and B, supplied by the user) are annealed to form a
double-stranded DNA fragment for ligation into the pGeneClip™ Vectors.
(Figures 1 and 4).
The hairpin oligonucleotides used in these systems are under 60bp in length.
Since detection of a 60bp insert is difficult using an agarose gel, we have
devised a method to allow detection of inserts by PstI digestion. The
pGeneClip™ Vectors contain a single PstI site. Insertion of the hairpin
oligonucleotides creates a second PstI site, and digestion with PstI will
yield two DNA fragments in the presence of an insert. PstI digestion of
pGeneClip™ Vectors that do not contain insert will result in linearization of
the vector (see Section 4.E).
Note: Two hairpin oligonucleotides must be ordered for each sequence tested.
Standard desalting of the oligonucleotides is required prior to use; gel
purification and 5´ phosphorylation are not required.
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Oligonucleotide A
Oligonucleotide A forms the template strand for cellular RNA polymerase II
and should contain the following elements:
• Overhang sequence. This four-nucleotide sequence (TCTC) is
complementary to the overhang of the pGeneClip™ Vectors and completes
the U1 promotor sequence.
• Hairpin sequence. Includes:
Target sequence. See Section 3.A. This sequence, in conjunction with its
reverse complement, forms the double-stranded portion of the cellular
siRNA hairpin.
Loop sequence. This sequence provides flexibility for RNA hairpin
formation within the cell. We have tested two different loop sequences
(CTTCCTGTCA and AAGTTCTCT) in our system and observed
comparable suppression levels with each. Other loop sequences reported in
the literature have not been tested (18).
Target sequence reverse complement. This sequence, in conjunction with the
target sequence, forms the double-stranded portion of the cellular RNA
hairpin.
• Additional CT residues. The additional CT residues are required for the
formation of the second PstI site upon ligation of the insert. These two
nucleotides also form the 3´ overhang required for successful siRNAs.
Hairpin Sequence
Loop
Reverse Complement
4711MA
Target
5´ TCTC
CT 3´
Figure 2. Structure of oligonucleotide A.
Oligonucleotide B
Oligonucleotide B has regions that are complementary to oligonucleotide A,
with additional sequences for ligation into the pGeneClip™ Vectors.
Oligonucleotide B should contain the following elements:
• Hairpin sequence complement. This sequence is complementary to, and in
the opposite orientation from, the hairpin sequence of oligonucleotide A.
Hairpin Sequence Complement
3´
GACGTC 5´
4712MA
• Overhang sequence. This sequence (GACGTC) is complementary to the
pGeneClip™ Vector overhang and contains an additional nucleotide to
form a PstI site upon successful ligation. This newly generated PstI site
allows easy detection of clones with inserts.
Figure 3. Structure of oligonucleotide B. Note that the sequence is represented
in the 3´ to 5´ orientation.
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An example of hairpin oligonucleotide sequences using the 19-nucleotide
target mRNA sequence, 5´ ggccuuucacuacuccuac 3´, is shown in Figure 4. Each
oligonucleotide in this example is 57 nucleotides in length.
4.
Cloning a Hairpin Insert into the pGeneClip™ Vectors
Materials to Be Supplied by the User
(Solution compositions are provided in Section 8.G)
• oligonucleotide A (see Section 3)
• oligonucleotide B (see Section 3)
• high-efficiency competent cells (‡1 × 108cfu/µg)
• 1.5ml polypropylene microcentrifuge tubes or 17 × 100mm polypropylene
tubes (Falcon Cat.# 2059)
• LB plates with ampicillin
• SOC medium
4.A. Oligonucleotide Dilution and Annealing
1. Dilute oligonucleotide A and oligonucleotide B in TE buffer or NucleaseFree Water to a final concentration of 1µg/µl.
2. Assemble the annealing reaction as described below.
oligonucleotide A (1µg/µl)
oligonucleotide B (1µg/µl)
Oligo Annealing Buffer
Total volume
2µl
2µl
46µl
50µl
The final concentration of each hairpin oligonucleotide is 40ng/µl.
3. Heat the annealing reaction at 90°C for 3 minutes.
4. Transfer to a 37°C water bath and incubate for 15 minutes.
5. The annealed hairpin oligonucleotides can be used immediately or stored
at –20°C for up to one month. If the annealed oligonucleotides are stored at
–20°C, thaw them at room temperature prior to use. Avoid thawing the
annealed oligonucleotides at temperatures above room temperature.
4.B. Ligation of a Hairpin Insert into the pGeneClip™ Vectors
The pGeneClip™ Vectors are provided as linearized vectors. No manipulation
of the vectors is required prior to ligation.
1. Dilute the annealed hairpin oligonucleotides from Section 4.A, Step 5, just
prior to assembling the ligation reaction as described below.
Note: Do not store the diluted oligonucleotides.
annealed hairpin oligonucleotides
Nuclease-Free Water
Total volume
5µl
45µl
50µl
The final concentration of each oligonucleotide is 4ng/µl.
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Target
Sequence
Loop
Target
Reverse
Complement
Target
Sequence
Loop
Target
Reverse
Complement
Pst I*
Figure 4. Example of hairpin oligonucleotide sequences.
*New Pst I site is generated to allow easy identification of positive clones.
Vector TCTCggcctttcactactcctacCTTCCTGTCAgtaggagtagtgaaaggccctGCAG
AGAGccggaaagtgatgaggatgGAAGGACAGTcatcctcatcactttccgggaCGTC
U1 Promoter
Transcription Start Site
Vector
10:36 AM
Hairpin Oligonucleotide Inserted into the pGeneClip™ Vector
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Overhang
3´
Oligonucleotide A 5´ TCTCggcctttcactactcctacCTTCCTGTCAgtaggagtagtgaaaggccct
Oligonucleotide B 3´
ccggaaagtgatgaggatgGAAGGACAGTcatcctcatcactttccgggaCGTC 5´
Overhang
Hairpin Oligonucleotide Sequence:
Target mRNA sequence: 5´ggccuuucacuacuccuac 3´
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2. Assemble the ligation reactions as described below. See Notes a and b.
Negative Control Standard
(Minus Insert)
Reaction
2X Rapid Ligation Buffer
5µl
5µl
pGeneClip™ Vector (50ng/µl)
1µl
1µl
annealed oligonucleotides A and B (4ng/µl each)
–
1µl
Nuclease-Free Water
3µl
2µl
T4 DNA Ligase (3 units/µl)
1µl
1µl
Total volume
10µl
10µl
Notes:
a. The 2X Rapid Ligation Buffer contains ATP, which degrades during
temperature fluctuations. Avoid multiple freeze-thaw cycles and
exposure to frequent temperature changes by making single-use aliquots
of the buffer after the buffer is thawed for the first time. Store the aliquots
at –20°C.
b. Vortex the 2X Rapid Ligation Buffer before each use.
3. Mix the reactions by pipetting. Incubate the reactions at room temperature
for 5 minutes. Alternatively, the reactions can be incubated for 1 hour at
room temperature or overnight at 4°C.
4.C. Transformation of E. coli with pGeneClip™ Vectors Containing Inserts
The ligation of fragments with a hairpin can be inefficient, so it is essential to
use competent cells with a transformation efficiency of ‡1 × 108cfu/µg DNA in
order to obtain a reasonable number of colonies. Transformation efficiency can
be confirmed by performing a control transformation reaction using a known
quantity of supercoiled plasmid DNA, typically 0.1ng, then calculating the
number of colony forming units per microgram of DNA.
We recommend using high-efficiency JM109 Competent Cells (Cat.# L2001).
Other host strains, such as DH5α™, may be used. If you are using competent
cells other than JM109 Competent Cells purchased from Promega, be sure to
follow the appropriate transformation protocol. Select transformants on
LB/ampicillin plates. For best results, do not use plates that are more than
1 month old.
Note: The use of plates containing IPTG and X-gal is not recommended.
Vectors containing insert may produce blue colonies. Therefore, blue/white
screening is not appropriate.
1. Prepare two LB/ampicillin plates for each ligation reaction and control transformation. Equilibrate the plates to room temperature prior to plating cells.
2. Remove the frozen, high-efficiency Competent Cells from –70°C storage
and place in an ice bath until just thawed (about 5 minutes). Mix the cells
by gently flicking the tube.
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3. For each ligation reaction and each transformation control, carefully
transfer 50µl of cells into a sterile 1.5ml microcentrifuge tube on ice.
Note: In our experience, using larger (17 × 100mm) polypropylene tubes
(e.g., Falcon Cat.# 2059) increases transformation efficiency. Tubes from
some manufacturers bind DNA, thereby decreasing the colony number,
and should be avoided.
4. Briefly centrifuge the tubes containing the ligation reactions to collect
contents at the bottom of the tube. Add 2µl of each ligation reaction to a
tube prepared in Step 3.
To perform a transformation control, add 0.1ng of supercoiled plasmid
DNA to one of the tubes prepared in Step 3.
Note: The pGeneClip™ Vectors are supplied linearized and are not
appropriate as a transformation control.
5. Gently flick the tubes to mix and place them on ice for 20 minutes.
6. Heat-shock the cells for 45–50 seconds in a water bath at 42°C. Do not shake.
7. Immediately return the tubes to ice for 2 minutes.
8. Add 950µl of room-temperature SOC medium to each tube. LB broth may
be substituted, but the number of colonies may be lower.
9. Incubate the tubes at 37°C with shaking (approximately 150rpm) for
1.5 hours.
10. Plate 50µl of each transformation onto duplicate LB/ampicillin plates. If a
higher number of colonies is desired, pellet the cells by centrifugation at
1,000 × g for 10 minutes, resuspend the cells in 200µl of SOC medium and
plate 50µl of cells on each of 2 plates.
11. Incubate the plates overnight (16–24 hours) at 37°C. In our experience, at
least 100 colonies per plate are routinely seen when using competent cells
that are 1 × 108cfu/µg DNA if 50µl is plated.
Notes:
1. The negative control ligation reaction allows determination of the number
of background colonies resulting from the GeneClip™ Vector alone. A
successful ligation reaction with insert typically yields at least 50 times
more colonies than the negative control ligation reaction. The efficiency of
ligation will depend upon the hairpin oligonucleotide sequence.
2. The transformation efficiency of the competent cells can be determined
using the following equation:
Equation for Transformation Efficiency (cfu/µg)
colonies on control plate
ng of supercoiled plasmid DNA plated
×
1 × 103ng
µg
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4.D. Purifying Recombinant Plasmid DNA
A standard plasmid miniprep procedure can be used for screening of inserts.
The miniprep process can be both laborious and time-consuming, particularly
when large numbers of minipreps are required. A convenient and reliable
method is the Wizard® Plus SV Minipreps DNA Purification System
(Cat.# A1330).
Plasmid purification protocols that reduce the amount of endotoxin in the
DNA preparation are preferred to minimize the toxic effects of endotoxins on
the cells during transfection.
4.E. Screening for Inserts Using PstI Digestion
The pGeneClip™ Vectors contain a single PstI site. A second PstI site is
created upon insertion of the hairpin oligonucleotides, and digestion with the
restriction enzyme PstI will yield two DNA fragments. PstI digestion of
pGeneClip™ Vectors that do not contain insert will result in linearization of
the vector (see Table 1).
Table 1. Sizes of PstI DNA Fragments Produced by Digestion of the
pGeneClip™ Vectors in the Absence and Presence of an Insert.
Vector
pGeneClip™ Basic Vector
pGeneClip™ Puromycin Vector
pGeneClip™ Hygromycin Vector
pGeneClip™ Neomycin Vector
pGeneClip™ hMGFP Vector
PstI Fragments of
Vector Without Insert
3,402bp
4,561bp
4,989bp
4,758bp
5,267bp
PstI Fragments of
Vector With Insert1
2,461bp + 991bp
3,209bp + 1,402bp
3,892bp + 1,147bp
3,817bp + 991bp
4,120bp + 1,197bp
1The
size of the larger PstI fragment may vary with the size of the hairpin oligonucleotide insert. The fragments sizes given here were determined using annealed hairpin
oligonucleotides with a double-stranded region of 50bp.
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Transfection of pGeneClip™ Vector Constructs in an siRNA
Suppression Assay
Once the annealed hairpin oligonucleotides are ligated to the appropriate
pGeneClip™ Vector, the resulting constructs can be used for transient
expression or for stable transfection. pGeneClip™ Vectors that contain a
selectable marker (pGeneClip™ Neomycin, Hygromycin or Puromycin Vectors)
can be used for stable expression of a pool of cells or individual clones.
Transfection of DNA into human cells may be mediated by cationic lipids,
calcium phosphate, DEAE-Dextran, polybrene-DMSO or electroporation.
5.A. Transient Transfection of the pGeneClip™ Vector Constructs
High transfection efficiency is essential for achieving substantial suppression
levels using a transient transfection approach. Prior to testing the inhibition,
optimize the transfection conditions for maximum efficiency in the system to
be tested (see below). The optimal conditions will vary with cell type,
transfection method used and the amount of DNA. When using the
pGeneClip™ Basic, Puromycin, Hygromycin or Neomycin Vectors,
optimization can be performed using a GFP reporter such as the Monster
Green® Fluorescent Protein phMGFP Vector (Cat.# E6421). The pGeneClip™
hMGFP Vector already contains the GFP reporter. The GFP reporter can also
be used to determine transfection efficiency for the assay. To test the
effectiveness of the pGeneClip™ Vector constructs (screening various
sequences for levels of inhibition), the use of a reporter, such as GFP, is highly
recommended. This control can be performed as a separate transfection to
determine the percentage of the cell population transfected or as a
cotransfection where flow cytometry can be used to sort GFP-positive cells.
The level of target RNA suppression in transfected cells can then be
determined by taking the transfection efficiency into account.
Obtaining maximum suppression requires optimizing specific assay
conditions. We have observed variations in suppression efficiency as a result
of the cell line, cell culture conditions, target sequence and transfection
conditions. Varying the amount of transfection reagent, amount of DNA and
cell density can influence transfection efficiency. Obtaining the highest
transfection efficiency with low toxicity is essential for maximizing the siRNA
interference (suppression) effect in a transient assay. Additionally, maintaining
healthy cell cultures is essential for this application. The key considerations are
discussed more fully below.
Cell Density (Confluence) at Transfection
The recommended cell density for most cell types at transfection is
approximately 30–50%; this level is lower than standard transfection
experiments where cells are plated at 50–70% confluency. The optimal cell
density should be determined for each cell type. Maintaining a dividing cell
culture is essential because effective gene suppression requires proliferating
cells. Continued proliferation and the need to passage cells should be
considered when determining the number of cells to plate.
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Cell Proliferation
Successful suppression of gene expression requires actively proliferating and
dividing cells, so maintaining healthy cell cultures is essential for this
application. It is essential to minimize the decrease in cell growth associated
with nonspecific transfection effects and to maintain cell culture under
subconfluent conditions to assure rapid cell division. We recommend using the
CellTiter-Glo® Luminescent Cell Viability Assay (Cat.# G7570) to monitor cell
viability and growth.
Time
The optimal time after transfection for analyzing interference effects must be
determined empirically by testing a range of incubation times. Typically little
inhibition is seen after 24 hours, but the maximal suppression time can vary
from 48 to 96 hours depending on the cells used and the experimental targets
tested.
5.B. Stable Transfection of the pGeneClip™ Puromycin, Hygromycin and
Neomycin Vector Constructs
Note: The pGeneClip™ Basic and hMGFP Vectors are not suitable for stable
transfection.
For stable expression, antibiotic selection must be applied following transfection.
Cell lines vary in the level of resistance to antibiotics, so the level of resistance of
a particular cell line must be tested before attempting stable selection of the cells.
A “kill curve” will determine the minimum concentration of the antibiotic
needed to kill nontransfected cells. The antibiotic concentration for selection will
vary depending on the cell type and the growth rate. In addition, cells that are
confluent are more resistant to antibiotics, so it is important to keep the cells
subconfluent. The typical effective ranges and lengths of time needed for
selection are given in Table 2.
Table 2. Typical Conditions for Selection of Stable Transfectants.
pGeneClip™ Vector
pGeneClip™ Puromycin Vector
pGeneClip™ Hygromycin Vector
pGeneClip™ Neomycin Vector
Effective
Antibiotic
Concentration
Puromycin
1–10µg/ml
Hygromycin 100–1,000µg/ml
G-418
100–1,000µg/ml
Time Needed
For Selection
2–7 days
3–10 days
3–14 days
For example, to generate a kill curve for G-418 selection, test G-418
concentrations of 0, 100, 200, 400, 600, 800 and 1,000µg/ml in the media to
determine the concentration that is toxic to nontransfected cells. The miminum
concentration of antibiotic that kills 100% of the cells should be used in
subsequent experiments.
Once the effective concentration of antibiotic has been determined, transfected
cells can be selected for resistance.
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1. Following transfection, seed the cells at a low cell density.
2. Apply antibiotic to the medium at the effective concentration determined
from the kill curve.
3. Prepare a control plate for all selection experiments by treating
nontransfected cells with antibiotic in medium under the experimental
conditions. This control plate will confirm whether the conditions of
antibiotic selection were sufficiently stringent to eliminate cells not
expressing the resistance gene.
4. Change the medium every 2–3 days until drug-resistant clones appear.
5. Once clones (or pools of cells) are selected, grow the cells in media
containing the antibiotic at a reduced antibiotic concentration, typically
25–50% of the level used during selection.
5.C. Quantitating siRNA Target Gene Suppression
Reduction of the targeted gene expression can be measured by 1) monitoring
phenotypic changes of the cell, 2) measuring changes in mRNA levels (e.g.,
using RT-PCR), or 3) detecting changes in protein level by immunocytochemistry or Western blot analysis (19–22). The suppression effect will
vary depending on the target, cell line and experimental conditions.
Controlling for nonspecific effects on other targets is very important. As a
negative control, cells can be transfected with either a nonspecific or scrambled
target sequence. This will show that suppression of gene expression is specific to
the expression of the hairpin siRNA target sequences. When suppression is
determined by Western analysis, positive controls for other genes (e.g., tubulin
or actin) should be included. Additional details can be found in reference 23.
Using the GeneClip™ hMGFP Vector, expression of hMGFP allows
normalization of target gene suppression to transfection efficiency.
Alternatively, the suppressive effects can be analyzed only in transfected cells by
separating those cells by FACS® analysis or by analyzing target gene
suppression at the individual cell level. Peak excitation of hMGFP occurs at
505nm, with a shoulder at 480nm, and peak emission occurs at 515nm. hMGFP
expression can be monitored by fluorescence microscopy using an excitation
filter of 470±20nm (470/40nm) and an emission filter of 515nm (long pass).
The psiCHECK™-1 and -2 Vectors (Cat.# C8011, C8021) can also be used to
measure target gene suppression. These Vectors enable the monitoring of
changes in expression of a target gene fused to a reporter, Renilla luciferase,
gene. In these vectors, the gene of interest is cloned into the multiple cloning
region located downstream of the Renilla translational stop codon. Initiation of
the RNAi process towards a gene of interest results in cleavage and
subsequent degradation of the fusion mRNA. Measurement of decreased
Renilla luciferase activity is a convenient indicator of RNAi effect (24).
Printed in USA.
Revised 1/14
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Page 15
6.
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10:36 AM
Page 16
Troubleshooting
Symptoms
Causes and Comments
Low number or
no colonies
Ligation reaction failed. Ligation reactions with
insert typically yield at least 50 times more
colonies than negative control reactions. If the
number of colonies is the same as the negative
control, this indicates a problem with the insert
or the ligation reaction.
The 2X Rapid Ligation Buffer contains ATP,
which degrades during temperature
fluctuations. Avoid multiple freeze-thaw cycles
by making single-use aliquots of the buffer. Use
a fresh vial of buffer.
Incorrect oligonucleotide sequences.
Oligonucleotide A must have an overhang of
TCTC, and oligonucleotide B must have an
overhang of CGTC. See Figure 4.
Improper dilution of the 2X Rapid Ligation
Buffer. The Rapid Ligation Buffer is provided at
a 2X concentration. Use 5µl in a 10µl reaction.
Ineffective transformation or poor-quality
competent cells. Perform a control transformation with supercoiled plasmid DNA to
ensure that the efficiency of the competent cells
is ‡1 × 108cfu/µg DNA (see Section 4.C). The
pGeneClip™ Vectors are supplied linearized
and are not appropriate as transformation
controls.
Oligonucleotides failed to anneal. Confirm
oligonucleotide sequences. Repeat annealing
step and use annealed oligonucleotides
immediately.
Insert not present in
pGeneClip™ Vector
Poor-quality oligonucleotides. Try HPLCpurified or gel-purified oligonucleotides.
Ligation reaction failed. Ligation reactions with
insert typically yield at least 50 times more
colonies than negative control reactions. If the
number of colonies is the same as the negative
control, this indicates a problem with the insert
or the ligation reaction.
The 2X Rapid Ligation Buffer contains ATP,
which degrades during temperature
fluctuations. Avoid multiple freeze-thaw cycles
by making single-use aliquots of the buffer. Use
a fresh vial of buffer.
Part# TM256
Page 16
Printed in USA.
Revised 1/14
1/28/2014
10:36 AM
Page 17
Symptoms
Causes and Comments
Insert not present in
pGeneClip™ Vector
(continued)
Incorrect oligonucleotide sequences.
Oligonucleotide A must have an overhang
of TCTC, and oligonucleotide B must have an
overhang of CGTC. See Figure 4.
No suppression or
low-level suppression of
target gene
Ineffective siRNA target sequence. Test at least
3–6 target sequences for each mRNA to identify
the sequences that result in the highest level of
suppression.
Time point not optimal. Assay cells at several
time points within 2–6 days after transfection to
determine the peak effect.
Low transfection efficiency. Use a GFP vector,
such as the Monster Green® Fluorescent Protein
phMGFP Vector (Cat.# E6421), to determine
transfection efficiency. If the efficiency is low,
optimize transfection conditions, as described in
Section 5.
Inadequate detection. If reduction of the
targeted gene is analyzed by immunocytochemistry or Western blot, check for antibody
specificity. Include controls (e.g., actin or
tubulin) in your analysis (20).
Cell growth or viability affected by specific
target sequence. Transfection of the vector may
affect cell proliferation when compared to
nontransfected cells. We recommend
monitoring cell viability and growth to account
for any changes in cell number during the
suppression assay.
7.
References
1. Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. Cell 101, 235–8.
2. Zamore, P.D. (2001) RNA interference: Listening to the sound of silence. Nat. Struct.
Biol. 8, 746–50.
3. Sharp, P.A. (2001) RNA interference–2001. Genes Dev. 15, 485–90.
4. Hutvagger, G. and Zamore, P.D. (2002) RNAi: Nature abhors a double strand. Curr.
Opin. Genet. Dev. 12, 225–32.
5. Dykxhoorn, D.M., Novina, C.D. and Sharp, P.A. (2003) Killing the messenger: Short
RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457–67.
6. Denli, A.M. and Hannon, G.J. (2003) RNAi: An ever-growing puzzle. Trends Biochem.
Sci. 28, 196–201.
Printed in USA.
Revised 1/14
Part# TM256
Page 17
7.
1/28/2014
10:36 AM
Page 18
References (continued)
7. Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) RNA interference is mediated by
21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200.
8. Yu, J-Y. et al. (2002) RNA interference by expression of short-interfering RNA and
hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047–52.
9. Sui, G. et al. (2002) A DNA vector-based RNAi technology to suppress gene
expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–20.
10. Brummelkamp, T.R., Bernards, R. and Agami, R. (2002) A system for stable
expression of short interfering RNAs in mammalian cells. Science 296, 550–3.
11. Novarino, G. et al. (2004) Involvement of the intracellular ion channel CLIC1 in
microglia-mediated b-amyloid-induced neurotoxicity. J. Neurosci. 24, 5322–30.
12. Cormack, B.P., Valdivia, R.H. and Falkow, S. (1996) FACS-optimized mutants of the
green fluorescent protein (GFP). Gene 173, 33–8.
13. Sorensen, T.U. et al. (1999) Safe sorting of GFP-transduced live cells for subsequent
culture using a modified FACS vantage. Cytometry 37, 284–90.
14. Galbraith, D.W. et al. (1999) Flow cytometric analysis and FACS sorting of cells based
on GFP accumulation. Methods Cell. Biol. 58, 315–41.
15. Khvorova, A. et al. (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell
115, 209–16.
16. Ui-Tei, K. et al. (2004) Guidelines for the selection of highly effective siRNA sequences
for mammalian and chick RNA interference. Nucl. Acid Res. 32, 936–48.
17. Vidugiriene, J. et al. (2004) The use of bioluminescent reporter genes for RNAi
optimization. Promega Notes 87, 2–6.
18. Elbashir, S.M. et al. (2002) Analysis of gene function in somatic mammalian cells
using small interfering RNAs. Methods 26, 199–213.
19. Xia, H. et al. (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotech.
20, 1006–10.
20. Huang, Y. et al. (2003) Erbin suppresses the MAP kinase pathway. J. Biol. Chem. 278,
1108–14.
21. Kullmann, M. et al. (2002) ELAV/Hu proteins inhibit p27 translation via an IRES
element in the p27 5´UTR. Genes Dev. 16, 3087–99.
22. Lang, V. et al. (2003) bTrCP-mediated proteolysis of NF-kB p105 requires
phosphorylation of p105 series 927 and 932. Mol. Cell. Biol. 23, 402–13.
23. Editorial (2003) Whither RNAi? Nature Cell Biol. 5, 489–90.
24. Kumar, R. et al. (2003) High-throughput selection of effective RNAi probes for gene
silencing. Genome Res. 13, 2333–40.
Part# TM256
Page 18
Printed in USA.
Revised 1/14
8.
1/28/2014
10:36 AM
Page 19
Appendix
8.A. pGeneClip™ Vector Maps and Sequence Reference Points
The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed
locations of the vector features and restriction enzyme sites are in relation to
base 1 (the T7 RNA polymerase transcription initiation site) and are numbered
as though the overhangs had been filled in and then ligated.
➞
T7
linearized pGeneClip™
Basic Vector
(3,402bp)
1 start
U1 promoter
Amp r
➞
Pst I 1432
G
C
A
G
SP6 529
4790MA
A
G
A
G
Figure 5. pGeneClip™ Basic Vector circle map and sequence reference points.
pGeneClip™ Basic Vector Sequence Reference Points
T7 RNA polymerase transcription initiation site
U1 promoter (human –392 to +1)
10bp spacer
U1 termination sequence
SP6 RNA polymerase promoter (–17 to +3)
SP6 RNA polymerase promoter binding site
Binding site of pUC/M13 Reverse Sequencing Primer
b-lactamase (Ampr) coding region
Binding site of pUC/M13 Forward Sequencing Primer
T7 RNA polymerase promoter (–17 to +3)
1
46–438
439–448
449–465
527–546
529–547
564–585
1724–2584
3336–3359
3386–3
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Page 20
➞
T7
Puromycin Vector
(4,561bp)
U1 promoter
Amp r
1 start
A
G
A
G
Puromycin
➞
SP6 529
4791MA
Pst I
Synthetic 1843
Polyadenylation
Signal
G
C
A
G
SV40 Enhancer/
Early Promoter
Figure 6. pGeneClip™ Puromycin Vector circle map and sequence reference
points.
pGeneClip™ Puromycin Vector Sequence Reference Points
10bp spacer
SV40 enhancer and early promoter
SV40 minimum origin of replication
Puromycin-N-acetyltransferase coding region
Synthetic polyadenylation signal
1
46–438
439–448
449–465
527–546
529–547
564–585
798–1216
1114–1179
1239–1838
1883–1931
2883–3743
4495–4518
4545–3
Part# TM256
Page 20
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Revised 1/14
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10:36 AM
Page 21
T7
Hygromycin Vector
U1 promoter
Amp r
1 start
(4,989bp)
A
G
A
G
Hygromycin
SP6 529
4789MA
Pst I
1588
➞
Synthetic
Polyadenylation
Signal
G
C
A
G
SV40 Enhancer/
Early Promoter
Figure 7. pGeneClip™ Hygromycin Vector circle map and sequence reference
points.
pGeneClip™ Hygromycin Vector Sequence Reference Points
10bp spacer
Hygromycin phosphotransferase coding region
1
46–438
439–448
449–465
527–546
529–547
564–585
798–1216
1114–1179
1251–2276
2311–2359
3311–4171
4923–4946
4973–3
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Page 22
➞
T7
Neomycin Vector
(4,758bp)
1 start
U1 promoter
Amp r
A
G
A
G
Neomycin
SP6 529
4788MA
Pst I
1432
➞
Synthetic
Polyadenylation
Signal
G
C
A
G
SV40 Enhancer/
Early Promoter
Figure 8. pGeneClip™ Neomycin Vector circle map and sequence reference
points.
pGeneClip™ Neomycin Vector Sequence Reference Points
10bp spacer
Neomycin phosphotransferase coding region
1
46–438
439–448
449–465
527–546
529–547
564–585
798–1216
1114–1179
1251–2045
2080–2128
3080–3940
4692–4715
4742–3
Part# TM256
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Page 23
➞
T7
1 start
U1 promoter
Amp r
hMGFP Vector
(5,267bp)
A
G
A
G
hMGFP
G
C
A
G
Pst I
1638
➞
CMV
Promoter
SP6 529
4792MA
Intron
Figure 9. pGeneClip™ hMGFP Vector circle map and sequence reference points.
pGeneClip™ hMGFP Vector Sequence Reference Points
10bp spacer
CMV enhancer/promoter
Chimeric intron
hMGFP open reading frame
1
46–438
439–448
449–465
527–546
529–547
564–585
801–1550
1690–1822
1880–2563
2589–2637
3589–4449
5201–5224
5250–3
Printed in USA.
Revised 1/14
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Page 24
8.B. pGeneClip™ Basic Vector Restriction Enzyme Sites
The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed locations
of the restriction enzyme sites are in relation to base 1 (the T7 RNA polymerase
transcription initiation site). The following restriction enzyme tables were constructed
using DNASTAR® sequence analysis software. Please note that we have not verified
this information by restriction digestion with each enzyme listed. The location given
specifies the 3´ end of the cut DNA (the base to the left of the cut site). For more
information on the cut sites of these enzymes, or if you identify a discrepancy,
please contact your local Promega Branch or Distributor. In the U.S., contact
Promega Technical Services at 800-356-9526. Vector sequences are also available in
the GenBank® database (GenBank®/EMBL Accession Number AY744385) and on the
Internet at: www.promega.com/vectors/
Table 3. Restriction Enzymes That Cut the pGeneClip™ Basic Vector 1–5 Times.
Enzyme
AatII
AccI
AcyI
AflIII
Alw26I
Alw44I
AlwNI
ApaI
AspHI
AvaI
AvaII
BamHI
BanI
BanII
BglI
BglII
BsaI
BsaAI
BsaHI
Bsp120I
BspHI
BssSI
BstXI
BstZI
Bsu36I
Cfr10I
ClaI
DdeI
# of Sites
1
2
2
2
2
2
2
1
4
2
4
1
3
4
2
1
1
1
2
1
2
2
1
1
1
2
1
5
Location
112
29, 459
109, 2334
502, 905
1858, 2634
1219, 2464
242, 1321
14
497, 1223, 2383, 2468
357, 487
50, 305, 1935, 2157
40
649, 1745, 3028
14, 358, 497, 3066
1917, 3235
432
1858
2991
109, 2334
10
1624, 2632
1078, 2461
506
468
46
1877, 3092
476
46, 1180, 1588, 1754,
2294
1663, 1682, 2374
2991
1013, 2946
Enzyme
EaeI
EagI
EarI
EclHKI
Eco52I
Eco81I
EcoICRI
EcoRI
EcoRV
FokI
# of Sites
4
1
3
1
1
1
1
1
1
5
FspI
HaeII
2
4
HgaI
5
HincII
HindII
HpaI
Hsp92I
MaeI
2
2
1
2
5
MluI
NaeI
NciI
NgoMIV
NotI
NsiI
NspI
DraI
3
PaeR7I
DraIII
1
Ppu10I
DrdI
2
PstI*
*A second PstI site is created upon ligation of an. insert.
1
1
3
1
1
1
1
1
1
1
Location
468, 744, 2185, 3372
468
789, 2592, 3280
1797
468
46
495
34
18
522, 1763, 1944,
2231, 3318
2019, 3242
783, 1153, 3142,
3150
84, 1016, 1593,
2323, 3208
30, 483
30, 483
483
109, 2334
23, 1400, 1652,
1987, 3142
502
3094
1285, 1980, 2331
3092
468
515
909
487
511
1432
Part# TM256
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Page 25
Table 3. Restriction Enzymes That Cut the pGeneClip™ Basic Vector 1–5 Times
(continued).
Enzyme
PvuI
PvuII
# of Sites
2
3
RsaI
SacI
SalI
ScaI
SinI
1
1
1
1
4
Location
2167, 3263
333, 729, 3292
2277
497
28
2277
50, 305, 1935, 2157
Enzyme
SspI
StyI
TfiI
VspI
XbaI
XhoI
XmnI
# of Sites
2
1
3
3
1
1
2
Location
2601, 2783
120
325, 740, 880
676, 735, 1969
22
487
277, 2396
Table 4. Restriction Enzymes That Do Not Cut the pGeneClip™ Basic Vector.
AccB7I
AccIII
Acc65I
AflII
AgeI
AscI
AvrII
BalI
BbeI
BbrPI
BbsI
BbuI
BclI
BlpI
Bpu1102I
BsaBI
BsaMI
BsmI
BspMI
BsrGI
BssHII
Bst1107I
Bst98I
BstEII
CspI
Csp45I
DraII
DsaI
Eco47III
Eco72I
EcoNI
EheI
FseI
HindIII
I-PpoI
KasI
KpnI
NarI
NcoI
NdeI
NheI
NruI
PacI
PflMI
PinAI
PmeI
PmlI
PpuMI
PshAI
Psp5II
PspAI
RsrII
SacII
SfiI
SgfI
SgrAI
SmaI
SnaBI
SpeI
SphI
SplI
SrfI
Sse8387I
StuI
SwaI
Tth111I
XcmI
XmaI
Table 5. Restriction Enzymes That Cut the pGeneClip™ Basic Vector 6 or More
Times.
AciI
AluI
BbvI
BsaOI
BsaJI
Bsp1286I
BsrI
BsrSI
Bst71I
BstOI
BstUI
CfoI
DpnI
DpnII
Fnu4HI
HaeIII
HhaI
HinfI
HpaII
HphI
Hsp92II
MaeII
MaeIII
MboI
MboII
MnlI
MseI
MspI
MspA1I
NdeII
NlaIII
NlaIV
PleI
Sau3AI
Sau96I
ScrFI
SfaNI
TaqI
Tru9I
XhoII
Note: The enzymes listed in boldface type are available from Promega.
Printed in USA.
Revised 1/14
Part# TM256
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Page 26
8.C. pGeneClip™ Puromycin Vector Restriction Enzyme Sites
transcription initiation site). The following restriction enzyme tables were constructed
Table 6. Restriction Enzymes That Cut the pGeneClip™ Puromycin Vector 1–5
Times.
Enzyme
AatII
AccI
Acc65I
AflIII
AgeI
Alw26I
Alw44I
AlwNI
ApaI
AspHI
# of Sites
3
3
1
2
1
3
2
2
1
5
AvaI
AvrII
BalI
BamHI
BbeI
BbuI
BglI
4
1
1
2
2
2
5
BglII
BsaI
BsaAI
Bsp120I
BspHI
BssHII
BssSI
BstEII
BstXI
BstZI
Bsu36I
Cfr10I
1
2
1
1
2
1
2
1
1
2
1
4
Location
112, 1285 1783
29, 459, 1348
845
502, 2063
1868
1713, 3017, 3793
2377, 3623
242, 2479
14
497, 1672, 2381,
3542, 3627
357, 487, 1288, 1849
1195
1614
40, 1844
1450, 1581
943, 1015
1148, 1455, 1586,
3076, 4394
432
1713, 3017
4150
10
2783, 3791
1694
2236, 3620
1373
506
468, 1687
46
1585, 1868, 3036,
4251
Enzyme
ClaI
CspI
Csp45I
DraI
# of Sites
1
1
1
4
DraIII
DrdI
DsaI
EagI
EarI
EclHKI
Eco52I
Eco81I
EcoICRI
EcoRI
EcoRV
EheI
FspI
HincII
HindII
HindIII
HpaI
KasI
KpnI
MluI
NaeI
NarI
NcoI
NgoMIV
NheI
NotI
NsiI
2
2
4
2
4
1
2
1
1
1
1
2
3
3
3
2
1
2
1
1
1
2
2
1
1
1
3
Location
476
1355
1933
1857, 2822, 2841,
3533
1810, 4150
2171, 4105
806, 1102, 1255, 1450
468, 1687
789, 1398, 3751, 4439
2956
468, 1687
46
495
34
18
1448, 1579
799, 3178, 4401
30, 483, 1349
30, 483, 1349
1211, 1942
483
1446, 1577
849
502
4253
1447, 1578
806, 1102
4251
1229
468
515, 945, 1017
Part# TM256
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Page 27
Table 6. Restriction Enzymes That Cut the pGeneClip™ Puromycin Vector 1–5
Times (continued).
Enzyme
NspI
PaeR7I
PinAI
PmeI
Ppu10I
PspAI
PstI*
PvuI
PvuII
RsaI
RsrII
SacI
SacII
SalI
ScaI
# of Sites
3
2
1
1
3
1
1
2
4
4
1
1
1
2
1
Location
943, 1015, 2067
487, 1849
1868
1857
511, 941, 1013
1288
1843
3326, 4422
333, 729, 871, 4451
847, 1248, 1297, 3436
1355
497
1453
28, 1347
3436
Enzyme
SfiI
SmaI
SphI
SplI
SspI
StuI
StyI
TfiI
Tth111I
VspI
XbaI
XhoI
XmaI
XmnI
.
*A second PstI site is created upon ligation of an insert.
# of Sites
1
1
2
1
2
2
5
4
1
3
2
2
1
2
Location
1148
1290
943, 1015
1295
3760, 3942
1194, 1570
120, 806, 1102, 1195,
1591
325, 740, 1217, 2038
1281
676, 735, 3128
22, 1862
487, 1849
1288
277, 3555
Table 7. Restriction Enzymes that Do Not Cut the pGeneClip™ Puromycin Vector.
AccB7I
AccIII
AflII
AscI
BbrPI
BbsI
BclI
BlpI
Bpu1102I
BsaBI
BsaMI
BsmI
BspMI
BsrGI
Bst1107I
Bst98I
DraII
Eco47III
Eco72I
EcoNI
FseI
I-PpoI
NdeI
NruI
PacI
PflMI
PmlI
PpuMI
PshAI
Psp5II
SgfI
SgrAI
SnaBI
SpeI
SrfI
Sse8387I
SwaI
XcmI
Table 8. Restriction Enzymes that Cut the pGeneClip™ Puromycin Vector 6 or
More Times.
AciI
AcyI
AluI
AvaII
BanI
BanII
BbvI
BsaOI
BsaHI
BsaJI
Bsp1286I
BsrI
BsrSI
Bst71I
BstOI
BstUI
CfoI
DdeI
DpnI
DpnII
EaeI
Fnu4HI
FokI
HaeII
HaeIII
HgaI
HhaI
HinfI
HpaII
HphI
Hsp92I
Hsp92II
MaeI
MaeII
MaeIII
MboI
MboII
MnlI
MseI
MspI
MspA1I
NciI
NdeII
NlaIII
NlaIV
PleI
Sau3AI
Sau96I
ScrFI
SfaNI
SinI
TaqI
Tru9I
XhoII
Printed in USA.
Revised 1/14
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8.D. pGeneClip™ Hygromycin Vector Restriction Enzyme Sites
transcription initiation site) and are numbered as though the overhangs had been
filled in and then ligated. The following restriction enzyme tables were constructed
Table 9. Restriction Enzymes That Cut the pGeneClip™ Hygromycin Vector 1–5
Times.
Enzyme # of Sites
AatII
2
AccI
2
AccIII
3
Acc65I
1
AcyI
4
AflIII
2
AgeI
1
Alw26I
2
Alw44I
4
AlwNI
2
ApaI
1
AvaI
4
AvrII
1
BamHI
1
BanI
5
BanII
BbuI
BglI
BglII
BsaI
BsaAI
BsaHI
Bsp120I
BspHI
BspMI
BssSI
BstXI
BstZI
Bsu36I
Cfr10I
ClaI
4
2
3
1
1
1
4
1
2
1
4
1
4
1
4
1
Location
112, 1278
29, 459
1472, 2009, 2145
845
109, 1275, 2243, 3921
502, 2491
2296
3445, 4221
1531, 1833, 2805, 4051
242, 2907
14
357, 487, 1240, 2277
1195
40
649, 845, 1825, 3332,
4615
14, 358, 497, 4653
943, 1015
1148, 3504, 4822
432
3445
4578
109, 1275, 2243, 3921
10
3211, 4219
1564
1351, 1830, 2664, 4048
506
468, 1456, 1621, 2191
46
1589, 2296, 3464, 4679
476
Enzyme # of Sites
CspI
1
Csp45I
1
DraI
4
DraIII
3
DrdI
4
DsaI
5
EagI
EarI
EclHKI
Eco52I
Eco81I
EcoICRI
EcoRI
EcoRV
FspI
HaeII
HincII
HindII
HindIII
HpaI
Hsp92I
KpnI
MluI
NaeI
NcoI
NdeI
NgoMIV
NheI
NotI
NsiI
NspI
4
3
2
4
1
1
2
1
3
4
3
3
2
1
4
1
1
1
3
1
1
1
1
3
3
Location
1659
2361
2285, 3250, 3269, 3961
1539, 1832, 4578
1755, 2136, 2599, 4533
806, 1102, 1603, 1959,
2028
468, 1456, 1621, 2191
789, 4179, 4867
1313, 3384
468, 1456, 1621, 2191
46
495
34, 1494
18
799, 3606, 4829
783, 2739, 4729, 4737
30, 483, 2087
30, 483, 2087
1211, 2370
483
109, 1275, 2243, 3921
849
502
4681
806, 1102, 1603
1701
4679
1229
468
515, 945, 1017
943, 1015, 2495
Part# TM256
Page 28
Printed in USA.
Revised 1/14
1/28/2014
10:36 AM
Page 29
Table 9. Restriction Enzymes That Cut the pGeneClip™ Hygromycin Vector 1–5
Times (continued).
Enzyme
PaeR7I
PinAI
PmeI
Ppu10I
PshAI
PspAI
PstI*
PvuI
# of Sites
2
1
1
3
1
1
1
4
PvuII
RsaI
RsrII
SacI
SacII
SalI
ScaI
4
4
1
1
1
1
2
Location
487, 2277
2296
2285
511, 941, 1013
1278
1240
1588
1237, 1615, 3754,
4850
333, 729, 871, 4879
847, 2168, 2221, 3864
1659
497
2031
28
2221, 3864
Enzyme # of Sites
SfiI
1
SgfI
2
SmaI
1
SphI
2
SrfI
1
SspI
2
StuI
1
StyI
5
Tth111I
VspI
XbaI
XhoI
XmaI
XmnI
1
3
2
2
1
2
Location
1148
1237, 1615
1242
943, 1015
1242
4188, 4370
1194
120, 806, 1102,
1195,1603
1755
676, 735, 3556
22, 2290
487, 2277
1240
277, 3983
. insert.
*A second PstI site is created upon ligation of an
Table 10. Restriction Enzymes that Do Not Cut the pGeneClip™ Hygromycin
Vector.
AccB7I
AflII
AscI
BalI
BbeI
BbrPI
BbsI
BclI
BlpI
Bpu1102I
BsaBI
BsaMI
BsmI
BsrGI
BssHII
Bst1107I
Bst98I
BstEII
DraII
Eco47III
Eco72I
EcoNI
EheI
FseI
I-PpoI
KasI
NarI
NruI
PacI
PflMI
PmlI
PpuMI
Psp5II
SgrAI
SnaBI
SpeI
SplI
Sse 8387I
SwaI
XcmI
Table 11. Restriction Enzymes that Cut the pGeneClip™ Hygromycin Vector 6 or
More Times.
AciI
AluI
AvaII
BbvI
BsaOI
BsaJI
Bsp1286I
BsrI
BsrSI
Bst71I
BstOI
BstUI
CfoI
DdeI
DpnI
DpnII
EaeI
Fnu4HI
FokI
HaeIII
HgaI
HhaI
HinfI
HpaII
HphI
Hsp92II
MaeI
MaeII
MaeIII
MboI
MboII
MnlI
MseI
MspI
MspA1I
NciI
NdeII
NlaIII
NlaIV
PleI
Sau3AI
Sau96I
ScrFI
SfaNI
SinI
TaqI
TfiI
Tru9I
XhoII
Printed in USA.
Revised 1/14
Part# TM256
Page 29
1/28/2014
10:36 AM
Page 30
8.E. pGeneClip™ Neomycin Vector Restriction Enzyme Sites
transcription initiation site). The following restriction enzyme tables were
constructed using DNASTAR® sequence analysis software. Please note that we have
not verified this information by restriction digestion with each enzyme listed. The
location given specifies the 3´ end of the cut DNA (the base to the left of the cut
site). For more information on the cut sites of these enzymes, or if you identify a
discrepancy, please contact your local Promega Branch or Distributor. In the U.S.,
contact Promega Technical Services at 800-356-9526. Vector sequences are also
available in the GenBank® database (GenBank®/EMBL Accession Number
AY745746) and on the Internet at: www.promega.com/vectors/
Table 12. Restriction Enzymes That Cut the pGeneClip™ Neomycin Vector 1–5
Times.
Enzyme # of Sites
AatII
1
AccI
2
Acc65I
1
AcyI
3
AflIII
2
AgeI
1
Alw26I
2
Alw44I
2
AlwNI
2
ApaI
1
AvaI
4
AvaII
5
AvrII
BalI
BamHI
BanII
1
1
1
5
BbeI
BbuI
BglI
BglII
BsaI
BsaAI
BsaHI
Bsp120I
BspHI
BspMI
BssHII
BssSI
BstXI
BstZI
1
3
3
1
1
2
3
1
2
2
1
3
1
2
Location
112
29, 459
845
109, 1379, 3690
502, 2260
2065
3214, 3990
2574, 3820
242, 2676
14
357, 487, 1240, 2046
50, 305, 1895, 3291,
3513
1195
1461
40
14, 358, 497, 1744,
4422
1382
943, 1015, 1784
1148, 3273, 4591
432
3214
1683, 4347
109, 1379, 3690
10
2980, 3988
1266, 1647
1776
1971, 2433, 3817
506
468, 1285
Enzyme # of Sites Location
Bsu36I
1
46
Cfr10I
5
1698, 1879, 2065, 3233,
4448
ClaI
1
476
CspI
1
1895
Csp45I
1
2130
DraI
4
2054, 3019, 3038, 3730
DraIII
1
4347
DrdI
3
1406, 2368, 4302
DsaI
3
806, 1102, 1811
EagI
2
468, 1285
EarI
5
789, 1723, 1933, 3948,
4636
EclHKI
1
3153
Eco52I
2
468, 1285
Eco81I
1
46
EcoICRI
1
495
EcoRI
1
34
EcoRV
1
18
EheI
1
1380
FspI
4
799, 1481, 3375, 4598
HaeII
5
783, 1382, 2508, 4498,
4506
HincII
2
30, 483
HindII
2
30, 483
HindIII
2
1211, 2139
HpaI
1
483
Hsp92I
3
109, 1379, 3690
KasI
1
1378
KpnI
1
849
MluI
1
502
NaeI
2
1881, 4450
Part# TM256
Page 30
Printed in USA.
Revised 1/14
1/28/2014
10:36 AM
Page 31
Table 12. Restriction Enzymes That Cut the pGeneClip™ Neomycin Vector 1–5
Times (continued).
Enzyme # of Sites
NarI
1
NcoI
3
NgoMIV
2
NheI
1
NotI
1
NsiI
3
NspI
4
PaeR7I
2
PinAI
1
PmeI
1
Ppu10I
3
PspAI
1
PstI*
1
PvuI
3
PvuII
5
Location
1379
806, 1102, 1811
1879, 4448
1229
468
515, 945, 1017
943, 1015, 1784, 2264
487, 2046
2065
2054
511, 941, 1013
1240
1432
1237, 3523, 4619
333, 729, 871, 1485,
4648
847, 1685, 3633
1895
497
Enzyme
SalI
ScaI
SfiI
SgfI
SinI
SmaI
SphI
SrfI
SspI
StuI
StyI
Tth111I
VspI
XbaI
RsaI
3
XhoI
RsrII
1
XmaI
SacI
1
XmnI
. insert.
*A second PstI site is created upon ligation of an
# of Sites
1
1
1
1
5
1
3
1
2
1
5
1
3
2
2
1
2
Location
28
3633
1148
1237
50, 305, 1895, 3291,
3513
1242
943, 1015, 1784
1242
3957, 4139
1194
120, 806, 1102, 1195,
1811
1497
676, 735, 3325
22, 2059
487, 2046
1240
277, 3752
Table 13. Restriction Enzymes that Do Not Cut the pGeneClip™ Neomycin Vector.
AccB7I
AccIII
AflII
AscI
BbrPI
BbsI
BclI
BlpI
Bpu1102I
BsaBI
BsaMI
BsmI
BsrGI
Bst1107I
Bst 98I
BstEII
DraII
Eco47III
Eco72I
EcoNI
FseI
I-PpoI
NdeI
NruI
PacI
PflMI
PmlI
PpuMI
PshAI
Psp5II
SacII
SgrAI
SnaBI
SpeI
SplI
Sse 8387I
SwaI
XcmI
Table 14. Restriction Enzymes that Cut the pGeneClip™ Neomycin Vector 6 or
More Times.
AciI
AluI
AspHI
BanI
BbvI
BsaOI
BsaJI
Bsp1286I
BsrI
BsrSI
Bst71I
BstOI
BstUI
CfoI
DdeI
DpnI
DpnII
EaeI
Fnu4HI
FokI
HaeIII
HgaI
HhaI
HinfI
HpaII
HphI
Hsp92II
MaeI
MaeII
MaeIII
MboI
MboII
MnlI
MseI
MspI
MspA1I
NciI
NdeII
NlaIII
NlaIV
PleI
Sau3AI
Sau96I
ScrFI
SfaNI
TaqI
TfiI
Tru9I
XhoII
Printed in USA.
Revised 1/14
Part# TM256
Page 31
1/28/2014
10:36 AM
Page 32
8.F. pGeneClip™ hMGFP Vector Restriction Enzyme Sites
The pGeneClip™ Vectors have overhangs of AGAG and GCAG. The listed
locations of the restriction enzyme sites are in relation to base 1 (the T7 RNA
polymerase transcription initiation site). The following restriction enzyme
tables were constructed using DNASTAR® sequence analysis software.
Please note that we have not verified this information by restriction digestion
with each enzyme listed. The location given specifies the 3´ end of the cut
DNA (the base to the left of the cut site). For more information on the cut sites
of these enzymes, or if you identify a discrepancy, please contact your local
Promega Branch or Distributor. In the U.S., contact Promega Technical Services
at 800-356-9526. Vector sequences are also available in the GenBank® database
(GenBank®/EMBL Accession Number AY744386) and on the Internet at:
www.promega.com/vectors/
Table 15. Restriction Enzymes That Cut the pGeneClip™ hMGFP Vector 1–5
Times.
Enzyme # of Sites Location
AatII
5
112, 1078, 1131 1214,
1400
AccB7I
1
2458
AccI
2
29, 459
AflII
2
1628, 1647
AflIII
2
502, 2769
AgeI
1
2574
Alw44I
2
3083, 4329
AlwNI
2
242, 3185
ApaI
1
14
AvaI
4
357, 487, 1862, 2129
BalI
2
810, 864
BamHI
1
40
BbeI
2
1929, 2022
BbsI
2
1761, 2071
BclI
1
1887
BglII
1
432
BsaI
2
1715, 3723
BsaAI
3
1293, 2329, 4856
Bsp120I
1
10
BspHI
2
3489, 4497
BspMI
1
1677
BsrGI
2
896, 2232
BssSI
3
2140, 2942, 4326
Bst98I
2
1628, 1647
BstEII
1
2069
BstXI
2
506, 2431
BstZI
1
468
Bsu36I
1
46
Cfr10I
4
2555, 2574, 3742, 4957
Enzyme # of Sites
ClaI
1
Csp45I
1
DdeI
5
DraI
DraIII
DrdI
DsaI
EagI
EarI
EclHKI
Eco52I
Eco81I
EcoICRI
EcoRI
EcoRV
EheI
FspI
HincII
HindII
HindIII
HpaI
I-PpoI
KasI
MluI
NaeI
NarI
NcoI
NdeI
NgoMIV
3
2
3
2
1
4
1
1
1
2
1
2
2
2
4
4
2
1
1
2
1
2
2
2
1
2
Location
476
2639
46, 3044, 3453, 3619,
4159
3528, 3547, 4239
2458, 4856
1617, 2877, 4811
1313, 1878
468
789, 797, 4457, 5145
3662
468
46
495, 1527
34
18, 1870
1927, 2020
3884, 5107
30, 483, 1477, 2257
30, 483, 1477, 2257
1556, 2648
483
1651
1925, 2018
502
2557, 4959
1926, 2019
1313, 1878
1187
2555, 4957
Part# TM256
Page 32
Printed in USA.
Revised 1/14
1/28/2014
10:36 AM
Page 33
Table 15. Restriction Enzymes That Cut the pGeneClip™ hMGFP Vector 1–5
Times (continued).
Enzyme
NheI
NotI
NsiI
NspI
PaeR7I
PflMI
PinAI
Ppu10I
PspAI
PstI*
PvuI
# of Sites
1
1
1
1
1
1
1
1
1
1
4
PvuII
SacI
SalI
3
2
1
Location
1851
468
515
2773
487
2458
2574
511
1862
1638
1464, 1859, 4032,
5128
333, 729, 5157
497, 1529
28
Enzyme
ScaI
SgfI
SmaI
SnaBI
SpeI
SspI
StyI
TfiI
Tth111I
VspI
XbaI
XhoI
XmaI
XmnI
# of Sites
1
2
1
1
1
4
4
4
1
4
2
1
1
2
Location
4142
1464, 1859
1864
1293
952
805, 852, 4466, 4648
120, 1313, 1878, 2094
325, 740, 2175, 2744
2252
676, 735, 960, 3834
22, 2568
487
1862
277, 4261
*A second PstI site is created upon ligation of an insert.
Table 16. Restriction Enzymes that Do Not Cut the pGeneClip™ hMGFP Vector.
AccIII
Acc65I
AscI
AvrII
BbrPI
BbuI
BlpI
Bpu1102I
BsaBI
BsaMI
BsmI
BssHII
Bst1107I
CspI
DraII
Eco47III
Eco72I
EcoNI
FseI
KpnI
NruI
PacI
PmeI
PmlI
PpuMI
PshAI
Psp5II
RsrII
SacII
SfiI
SgrAI
SphI
SplI
SrfI
Sse8387I
StuI
SwaI
XcmI
Table 17. Restriction Enzymes that Cut the pGeneClip™ hMGFP Vector 6 or More
Times.
AciI
AcyI
AluI
Alw26I
AspHI
AvaII
BanI
BanII
BbvI
BglI
BsaOI
BsaHI
BsaJI
Bsp1286I
BsrI
BsrSI
Bst71I
BstOI
BstUI
CfoI
DpnI
DpnII
EaeI
Fnu4HI
FokI
HaeII
HaeIII
HgaI
HhaI
HinfI
HpaII
HphI
Hsp92I
Hsp92II
MaeI
MaeII
MaeIII
MboI
MboII
MnlI
MseI
MspI
MspA1I
NciI
NdeII
NlaIII
NlaIV
PleI
RsaI
Sau3AI
Sau96I
ScrFI
SfaNI
SinI
TaqI
Tru9I
XhoII
Printed in USA.
Revised 1/14
Part# TM256
Page 33
1/28/2014
10:36 AM
Page 34
8.G. Composition of Buffers and Solutions
LB plates with ampicillin
Add 15g agar to 1 liter of LB medium.
Autoclave. Allow the medium to cool
to 50°C before adding ampicillin to a
final concentration of 100µg/ml. Pour
30–35ml of medium into 85mm petri
dishes. Let the agar harden. Store at
4°C for up to 1 month or at room
temperature for up to 1 week.
2M Mg2+ stock
20.33g MgCl2 • 6H2O
24.65g MgSO4 • 7H2O
Add distilled water to 100ml. Filter
sterilize.
2X Rapid Ligation Buffer (provided)
60mM
20mM
20mM
2mM
10%
Tris-HCl (pH 7.8)
MgCl2
DTT
ATP
polyethylene glycol
(MW8000, ACS Grade)
Store in single-use aliquots at –20°C.
Avoid multiple freeze-thaw cycles.
Oligo Annealing Buffer (provided)
60mM
1.5M
60mM
10mM
Tris-HCl (pH 7.5)
NaCl
MgCl2
DTT
SOC medium (100ml)
Bacto®-tryptone
Bacto®-yeast extract
1M NaCl
1M KCl
2M Mg2+ stock, filtersterilized
1ml 2M glucose, filter-sterilized
2.0g
0.5g
1ml
0.25ml
1ml
Add Bacto®-tryptone, Bacto®-yeast
extract, NaCl and KCl to 97ml distilled
water. Stir to dissolve. Autoclave and
cool to room temperature. Add 2M
Mg2+ stock and 2M glucose, each to a
final concentration of 20mM. Bring the
volume to 100ml with sterile, distilled
water. The final pH should be 7.0.
TE buffer
10mM Tris-HCl (pH 8.0)
1mM EDTA
8.H. Related Products
Product
PstI
Size
3,000 units
15,000 units
Cat.#
R6111
R6115
Size
50 × 20µl reactions
Cat.#
P1700
RNA Production System
Product
T7 RiboMAX™ Express RNAi System
Part# TM256
Page 34
Printed in USA.
Revised 1/14
1/28/2014
10:36 AM
Page 35
RNAi Vector Systems
Product
psiCHECK™ 1 Vector
psiCHECK™ 2 Vector
Size
20µg
20µg
Cat.#
C8011
C8021
Size
100 assays
1,000 assays
20µg
Cat.#
E2810
E2820
E6421
Size
10ml
10 × 10ml
100ml
10 × 100ml
Cat.#
G7570
G7571
G7572
G7573
Size
50 preps
250 preps
Cat.#
A1330
A1460
50 preps
250 preps
A1340
A1470
Reporter Assay Systems
Product
Renilla Luciferase Assay System
Monster Green® Fluorescent Protein phMGFP Vector
Cell Proliferation Assay
Product
CellTiter-Glo® Luminescent Cell Viability Assay
Plasmid DNA Purification
Product
Wizard® Plus SV Minipreps DNA Purification System
Wizard® Plus SV Minipreps DNA Purification System
+ Vacuum Adapters
Printed in USA.
Revised 1/14
Part# TM256
Page 35
1/28/2014
10:36 AM
Page 36
(a)This product is sold solely for use for research purposes in fields other than plants. This product is not transferable. If the
purchaser is not willing to accept the conditions of this label license, Promega is willing to accept the return of the product
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patents for use of ddRNAi as a therapeutic agent or as a method to treat/prevent human disease, please contact Benitec at
[email protected]. For the use of ddRNAi in other fields, please contact CSIRO at: www.pi.csiro.au/RNAi.
(b)This product is covered under license from Carnegie Institution of Washington under U.S. Pat. Nos. 6,506,559, 7,538,095,
7,560,438, Australian Pat. No. 743798 and other patents pending. Commercial use of this product will require a separate
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(c)Patent Pending.
(d)Certain applications of this product may require licenses from others.
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this agreement shall be governed under the laws of the State of Wisconsin, USA.
(f)The gene encoding Monster Green® Fluorescent Protein is exclusively licensed to Promega Corporation under U.S. Pat. Nos.
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Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more
information.
CellTiter-Glo, Monster Green and Wizard are registered trademarks of Promega Corporation. GeneClip, psiCHECK and
RiboMAX are trademarks of Promega Corporation.
Bacto is a registered trademark of Difco Laboratories, Detroit, Michigan. DH5α is a trademark of Life Technologies, Inc.
DNASTAR is a registered trademark of DNASTAR, Inc. GenBank is a registered trademark of US Dept of Health and Human
Services.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more
information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the
most up-to-date information on Promega products.
Part# TM256
Page 36
Printed in USA.
Revised 1/14

GeneClip™ U1 Hairpin Cloning Systems InstrucƟ ons for use of Products

Transcription

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