pAM_gRNA_CTCF Vector
Catalog No.: 53123
Format:  10 µg
Description
The pAM_gRNA_CTCF Vector is designed for use in combination with Active Motif’s pAM_dCas9 Vector (Catalog No. 53122) and Active
Motif’s enChIP Kit (Catalog No. 53125). The enChIP method relies on the CRISPR/Cas9 system to direct a guide RNA (gRNA) to a specific genomic locus for immunoprecipitation. The gRNA is designed as the complementary sequence of the desired target locus. An enzymaticallyinactive form of the Streptococcus pyogenes Cas9 protein, which contains Active Motif’s unique AM-tag sequence, is co-transfected withthe gRNA. Following expression, the gRNA directs the dCas9 protein to its target sequence, immediately upstream of a Protospacer Adjacent Motif (PAM) (5´- NGG). Recognition of a PAM site leads to unwinding of the DNA and formation of an RNA-DNA heteroduplex. Cells
are then formaldehyde fixed and chromatin is prepared. An antibody directed against the AM-tag is used to enrich for genomic sequences
bound by the gRNA/dCas9 complex. DNA can be analyzed by qPCR or NGS to identify the enriched genomic regions. The pAM_gRNA_CTCF Vector can be used as a positive control for the enChIP Kit. It contains a gRNA sequence corresponding to a CTCF binding site on
chromosome 19 (5,804,115-5,804,209) which has been cloned into the pAM_gRNA empty vector (Catalog No. 53121). The pAM_gRNA_CTCF
vector should be used with the dual vector transfection protocol.
Contents• 10 µg of pAM_gRNA_CTCF Vector provided at a concentration of 100 ng/µl.
pAM_gRNA_CTCF Vector Features and Circle MapThe following features are present in the pAM_gRNA_CTCF Vector based on nucleotide sequence.
ColE1-derived plasmid origin of replication
182-770
NeomycinR/KanamycinR coding region1489-2283
M13 forward primer
3071-3088
U6 promoter
3685-3925
CTCF binding site3935-3954
gRNA scaffold
3955-4030
Sp6 promoter
4062-4080
M13 reverse primer
4098-4114
Note:  The pAM_gRNA_CTCF Vector must be used in
combination with Active Motif’s pAM_dCas9 Vector
for dual transfection and expression.

Quality Control
Plasmid construct has been confirmed by restriction analysis and sequence verified. For the complete pAM_gRNA_CTCF Vector sequence,
please visit the Documents tab at www.activemotif.com/enchip.
Shipping & Storage
Products are shipped on dry ice.
Resuspended DNA is stable for 6 months when stored at -20°C. Avoid repeated freeze/thaw cycles.

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

Transfection of gRNA and dCas9 expression plasmids
Cells can be transiently transfected with the pAM_gRNA_CTCF and pAM_dCas9 expression plasmids. The following protocol provides
recommendations for transfection using FuGENE® HD Transfection Reagent (Catalog No. 32042). Optimization may be required for eachcell line tested.
To determine the efficiency of the transfection, set up a duplicate transfection for each cell type used. Cell lysates can be prepared fromthe duplicate transfection reaction using Active Motif’s Nuclear Extract Kit (Catalog No. 40010) for analysis by Western blot. Use theAM-Tag polyclonal antibody (Catalog No. 61677) at a 1:250 - 1:1,000 dilution for detection of the AM-tagged dCas9 protein. If the tagged
protein is not detected, continue to optimize transfection conditions.
Calculate the number of transfections you will perform. Small scale reactions provide enough chromatin to perform one enChIP reaction. Large scale reactions provide enough chromatin to perform 5 enChIP reactions. We recommend running a no gRNA negative control
reaction (empty pAM_dCas9 vector, Catalog No. 53122) for each cell line tested.
1.

Seed cells in either a 6-well plate (small scale) or 10cm dish (large scale) using the appropriate growth medium. Incubate in a humidified incubator for 24 hours. Cells should be 70-80% confluent at the time of transfection.
Small Scale Cell Culture(1 enChIP rxn)

Large Scale Cell Culture(5 enChIP rxns)

Dual Vector

Dual Vector

Cell culture plate

6-well plate

10 cm dishCell seeding density•

55 x 10 cells

3 x 106 cells

Growth medium

10 ml

20 ml

*These conditions were established for cells with doubling times of 14-18 hours. Cell seeding densities may need to be optimized to ensure that cells are ~80% confluent at the time of transfection.

2. Prepare a separate microcentrifuge tube for each transfection reaction. To each tube add the recommended amount of DNA andOpti-MEM according to the table below.

Positive control
CTCF gRNANo gRNA
negative control

Small Scale Cell Culture

Large Scale Cell Culture

Dual  Vector

Dual  Vector

gRNA_CTCF vector

1 µg

2 µg

dCas9 vector

1 µg

2 µg

Opti-MEM

Up to 275 µl

Up to 550 µlgRNA_CTCF vector––dCas9 vector

1 µg

2 µg

Opti-MEM

Up to 275 µl

Up to 550 µl

3. Add FuGENE HD Transfection reagent drop wise directly to the DNA/media mixture. Do not allow FuGENE to come directly in contact with the plastic from the tube. Mix the solution by pipetting up and down and incubate at room temperature for 30 minutes.
Small scale cell culture:  Add 6 µl FuGENE to each transfection reaction.
Large scale cell culture:  Add 12 µl FuGENE to each transfection reaction.
4. Add the entire DNA/media/FuGENE mixture drop wise to each cell culture plate. Incubate on a shaking platform at 100 rpm for 2
minutes to evenly distribute the transfection mixture.
5. Return plate to humidified incubator for 24 hours.
6. 24 hours post-transfection, passage cells.
Small scale cell culture:  Transfer cells in each well of a 6-well plate to a 10 cm dish.
Large scale cell culture:  Transfer cells from a 10 cm dish to a 15 cm dish.
7. Return plate to humidified incubator for 24 hours.
8. Transfected cells are now ready to be processed for chromatin fixation using the enChIP Kit (Catalog No. 53125). If duplicate reactions were performed, cell lysates can be prepared using Active Motif’s Nuclear Extraction Kit (Catalog No. 40010) for Western blot
analysis of transfection efficiency using the AM-Tag polyclonal antibody (Catalog No. 61677) to recognize the tagged dCas9 protein.
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
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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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France Free Phone: 0800 90 99 79Germany Free Phone:
0800 181 99 10
Telephone:+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
E-mail:eurotech@activemotif.com
Active Motif Japan
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2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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Shanghai, 201315, China
Telephone: (86)-21-20926090
Hotline:400-018-8123
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North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

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