CCNE1 KO cell line available now. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control available. Knockout.
Images
Key facts
- Cell type
HCT116
- Species or organism
Human
- Tissue
Colon
- Form
Liquid
- Knockout validation
Next Generation Sequencing, Western blot
Knockout.
Alternative names
CCNE, CCNE1_HUMAN, Cyclin E1, Cyclin Es, Cyclin Et, CyclinE, G1/S specific cyclin E, G1/S-specific cyclin-E1, cyclin E variant ex5del, cyclin E variant ex7del
Recommended products
CCNE1 KO cell line available now. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control available. Knockout.
Alternative names
CCNE, CCNE1_HUMAN, Cyclin E1, Cyclin Es, Cyclin Et, CyclinE, G1/S specific cyclin E, G1/S-specific cyclin-E1, cyclin E variant ex5del, cyclin E variant ex7del
Key facts
- Cell type
HCT116
- Species or organism
Human
- Tissue
Colon
- Form
Liquid
- Knockout validation
Next Generation Sequencing, Western blot
- Mutation description
Knockout.
- Disease
Carcinoma
- Concentration
Properties
- Gene name
CCNE1
- Gene editing type
Knockout
- Gene editing method
CRISPR technology
- Knockout validation
Next Generation Sequencing, Western blot
Quality control
- STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
EU: 1 US: 1
- Adherent/suspension
Adherent
- Gender
Male
Handling procedures
- Initial handling guidelines
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.- Subculture guidelines
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
- Culture medium
McCoY5a + 10% FBS
- Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
Dry Ice
- Appropriate long-term storage conditions
-196°C
Notes
Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein. Should only heterozygous edits be achieved, you will be notified of the outcome and be asked to confirm whether the cell line is acceptable. All clones will be accompanied with DNA sequencing data, and the mutation description.
Recommended control: Human wild-type HCT116 cell line (Human wild-type HCT116 cell line ab288559). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Product promise
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
3 product images
Western blot - Human CCNE1 knockout HCT116 cell line (ab286494)
Western blot: Anti-CCNE1 antibody [EPR194] (Anti-Cyclin E1 antibody [EPR194] ab133266) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Cyclin E1 antibody [EPR194] ab133266 was shown to bind specifically to CCNE1. A band was observed at 45 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CCNE1 knockout cell line. To generate this image, wild-type and CCNE1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Cyclin E1 antibody [EPR194] (Anti-Cyclin E1 antibody [EPR194] ab133266) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: CCNE1 knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type MCF7 Human wild-type MCF7 cell line ab288560 cell lysate at 20 µg
Lane 4: CCNE1 knockout MCF7 Human CCNE1 knockout MCF7 cell line ab286303 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 45 kDa
Western blot - Human CCNE1 knockout HCT116 cell line (ab286494)
Western blot: Anti-CCNE1 antibody [EPR26497-64] (Anti-Cyclin E1 antibody [EP435E] ab33911) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-Cyclin E1 antibody [EP435E] ab33911 was shown to bind specifically to CCNE1. A band was observed at 45 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CCNE1 knockout cell line. To generate this image, wild-type and CCNE1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Cyclin E1 antibody [EP435E] (Anti-Cyclin E1 antibody [EP435E] ab33911) at 1/1000 dilution
Lane 1: Wild-type HCT 116 cell lysate at 20 µg
Lane 2: CCNE1 knockout HCT 116 cell lysate at 20 µg
Lane 3: Wild-type MCF7 Human wild-type MCF7 cell line ab288560 cell lysate at 20 µg
Lane 4: CCNE1 knockout MCF7 Human CCNE1 knockout MCF7 cell line ab286303 cell lysate at 20 µg
SecondaryAll lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 45 kDa
Next Generation Sequencing - Human CCNE1 knockout HCT116 cell line (ab286494)
1bp insertion after Lys79, 3bp deletion downstream (allele 1); 1bp deletion after Lys79, 1bp insertion downstream (allele 2); 1bp insertion after Lys79, 1 bp insertion downstream (allele 3)
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