pAM_dCas9_CTCF Vector
Catalog No.: 53124
Format:  10 µg
Description
The pAM_dCas9_CTCF Vector is designed for use with Active Motif’s enChIP Kit (Catalog No. 53125). The enChIP method relies on theCRISPR/Cas9 system to direct a guide RNA (gRNA) to a specific genomic locus for immunoprecipitation. The gRNA is designed as thecomplementary sequence of the desired target locus. An enzymatically inactive form of the Streptococcus pyogenes Cas9 protein, whichcontains Active Motif’s unique AM-tag sequence, is co-expressed with the gRNA. 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 DNAand 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_dCas9_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_dCas9 emptyvector (Catalog No. 53122). The pAM_dCas9_CTCF vector should be used with the single vector transfection protocol.
Contents• 10 µg of pAM_dCas9_CTCF Vector provided at a concentration of 100 ng/µl.
pAM_dCas9_CTCF Vector Features and Circle Map
The following features are present in the pAM_dCas9_CTCF Vector based on nucleotide sequence.
dCas9 reverse primer24-43
CMV promoter
430-633
dCas9 coding region748-4851
AM-Tag4888-4980
Herpes simplex virus TK poly (A) signal5079-5127
f1 origin of replication
5329-5757
NeomycinR/KanamycinR coding region
6167-6961
SV40 polyadenylation signal7137-7258
ColE1-derived plasmid origin of replication 7709-8297
b-lactamase (Ampr) coding region
8468-9328
dCas9 forward primer
9538-9557
U6 promoter
9699-9939
CTCF binding site9949-9967
gRNA scaffold
9969-10044
Note:  The pAM_dCas9 empty vector (Catalog No.
53122) is available separately and is recommended foruse in combination with pAM_dCas9_CTCF as a nogRNA negative control.
Quality Control
Plasmid construct has been confirmed by restriction analysis and sequence verified. For the complete pAM_dCas9_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

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Transfection of pAM_dCas9_CTCF expression plasmid
Cells can be transiently transfected with the pAM_dCas9_CTCF expression plasmid. The following protocol provides recommendations
for transfection using FuGENE® HD Transfection Reagent (Catalog No. 32042). Optimization may be required for each cell 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)

Single Vector

Single 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.
Small Scale Cell Culture

Large Scale Cell Culture

Single Vector

Single Vector

Positive control
CTCF gRNA(dCas9_CTCF) vector

1 µg

2 µg

Opti-MEM

Up to 275 µl

Up to 550 µl

No gRNA
negative control

dCas9 vector

1 µg

2 µg

Opti-MEM

Up to 275 µl

Up to 550 µl3. 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 2minutes 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). Alternatively, 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.
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Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the numbers listed below.
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Telephone: +81 3 5225 3638
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+81 3 5261 8733
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North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com