pAM_1C Empty Vector
Catalog No.: 53023
Format:  20 µg
Description
The pAM_1C Empty Vector is designed for use with Active Motif’s Tag-ChIP-IT® Kit (Catalog No. 53022). The DNA sequence of interest iscloned into the pAM_1C vector using the HindIII restriction site to append the AM-tag sequence to the carboxy-terminus of the protein ofinterest. Alternatively, the AM-tag coding region can be removed and transferred into other protein expression vectors as a C-terminus tag.
pAM_1C contains the Human Beta-actin promoter (ACTB) which provides constitutive high-level protein expression in mammalian systems.
The vector may be used for transient transfections, or puromycin selection can be applied to select for stable gene expression. Westernblot analysis with the AM-Tag antibody (Catalog No. 61677) can confirm tagged-protein expression. Cells expressing the fusion protein are
ready for use in the Tag-ChIP-IT Kit.
Contents• 20 µg of pAM_1C Empty Vector provided as lyophilized DNA.
• 250 pmol pAM Forward primer for DNA sequencing provided as lyophilized DNA.
• 250 pmol pAM Reverse primer for DNA sequencing provided as lyophilized DNA.
Resuspend plasmid to a concentration of 1 µg/µl in sterile H2O. Store reconstituted DNA at -20°C. Resuspend DNA sequencing primers
to a concentration of 5 pmol/µl  in sterile H2O or TE Buffer (pH 7.5-8.0). Store reconstituted primers at -20°C.
pAM_1C Empty Vector Features and Circle Map
The following features are present in the pAM_1C Empty Vector based on nucleotide sequence.
SV40 poly(A) signal
pAM Reverse primer
Puromycin coding region
SV40 poly(A) signal
ColE1-derived plasmid origin of replicationb-lactamase (Ampr) coding region
Synthetic polyadenylation signal
RNA pol II transcriptional pause site
ACTB promoter
pAM Forward primer
Universal cloning site (HindIII)
AM-tag40-161
247-269543-1142
1149-1283
1686-2274
2474-3334
3439-3487
3501-3592
3627-4626
4507-4526
4644
4650-4751

Note:  The insert must contain an in-frame ATG codon
for translation initiation.
pAM_1C contains a TAG stop codon immediately
following the AM-tag sequence.

Quality Control
Plasmid construct has been confirmed by restriction analysis and sequence verified.
Shipping & Storage
Products are shipped at room temperature.
Lyophilized DNA is stable for 12 months when stored at -20°C.
Resuspended DNA is stable for 6 months when stored at -20°C. Avoid repeated freeze/thaw cycles.
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GENERAL PRODUCT USE
pAM_1C is an expression plasmid containing the constitutive mammalian ACTB promoter, a HindIII restriction site for cloning and a
C-terminal AM-tag. This vector may be used for cloning and expression of the gene of interest or as a template for cloning the AM-tag intoanother expression vector.
The following guidelines are provided for cloning:
1. Design a fusion protein construct containing the AM-tag in-frame with the C-terminus of the protein of interest. We recommend
the use of directional cloning methods such as InFusion® (Clontech) or Gibson Assembly® (New England BioLabs). Follow the manufacturer’s recommendations for cloning.
Alternatively, the AM-tag sequence can be cloned into the expression vector of choice for your protein of interest. Ensure that theopen reading frame of interest begins with the ATG start codon, that the AM-tag remains in-frame with the C-terminus and that astop codon is present. We do not recommend the use of the AM-tag at the N-terminus as this may significantly reduce expression
efficiency. If you require a N-terminal tag, please contact Active Motif’s technical support.
2. Perform transformations of the AM-tag fusion protein construct into competent E.coli. We recommend the use of Active Motif’s
Rapid Trans TAM1 Competent cells (Catalog No. 11096) or other commonly used E.coli strains such as DH5a. Use the appropriate antibiotic selection for the cloning vector chosen (e.g. pAM_1C contains the ampicillin resistance gene).
3. Select 2-4 colonies for each construct and isolate the plasmid DNA. Sequence validate each plasmid to confirm the correct open
reading frame sequence. We suggest preparing a glycerol stock for each candidate construct.
4. Determine the amount of DNA needed for transfection. If necessary, prepare additional DNA. Ensure that the DNA is free of nucleases or other contaminants prior to transfection. Quantify the DNA.
Following sequence validation of the AM-tag cloning vector, cells can be transiently transfected or selected to make stable cells expressing the AM-tagged protein of interest. Chromatin will be prepared from these cells for analysis in the Tag-ChIP-IT Kit (Catalog No. 53022).
Successful chromatin immunoprecipitation depends on the efficiency of the transfection and the expression levels of the AM-tag fusion
protein of interest. It is recommended to optimize the transfection and expression of tagged protein prior to performing ChIP.
Once transfection conditions have been optimized, calculate the number of chromatin preparations you will require and determine thenumber of ChIP reactions you plan to perform on each chromatin preparation. Be sure to include the appropriate control ChIP reactions in your calculations. We recommend using untransfected cells (or a non-expressing cell line) as an estimate of the background. It is
important to transfect enough cells to yield the necessary 20-30 µg of chromatin per immunoprecipitation reaction.
We recommend using FuGENE® HD Transfection Reagent (Catalog No. 32042) with the pAM_1C Vector at a ratio of 3:1 (µl FuGENE : µgDNA vector). Optimization may be required for each cell line and construct tested.
The following guidelines are provided for transfection:
1. In a 100 mm dish, seed 2.2 x 106 cells per plate in the appropriate growth medium. Incubate in a humidified incubator for 24 hours.
(Optional)  Prepare transfection reactions to verify expression of the AM-tagged fusion protein. We suggest performing a timecourse experiment to evaluate tagged-protein expression levels at 24, 48 and 72 hours post-transfection to identify the optimal
harvest time point for the protein of interest. Cell lysates can be prepared from these transfection reactions for analysis by Westernblot. Use the AbFlex™ AM-Tag antibody (Catalog No. 91111) at a 1:500 - 1:1000 dilution for detection of the tagged protein as compared
to untransfected cells.
2. Prepare a separate microcentrifuge tube for each transfection reaction. Add 10 µg AM-tag construct to Opti-MEM media in a final
volume of 550 µl.
3. Add 30 µl FuGENE HD Transfection Reagent drop wise directly to the media/DNA mixture. Do not allow FuGENE to come directly
into contact with the plastic. Mix the solution by pipetting up and down and incubate at room temperature for 15-30 minutes.
4. Add 580 µl of the media/DNA/FuGENE mixture drop wise to each 100 mm dish. Gently swirl dish or incubate on a shaker at 100 rpm
for 2 minutes to evenly distribute transfection mixture.
5. Return dish to humidified incubator for 24-72 hours before proceeding with the chromatin preparation. If desired, treat cells withcompounds.
6. Refer to the Tag-ChIP-IT manual to prepare chromatin for use in ChIP.
North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the numbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USAToll Free: 877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
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B-1330 La Hulpe, Belgium
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0800 169 31 47
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0800 181 99 10
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Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
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+81 3 5261 8733
E-mail:japantech@activemotif.com
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Telephone: (86)-21-20926090
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North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com