lentiCRISPR v2

CRISPR Mouse - Deletion NSC34 PGRMC1

Experiment
CRISPR Mouse - Deletion NSC34 PGRMC1
Product
lentiCRISPR v2 from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
There is no need to perform a negative control golden-gate reaction (without
insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

2.5. Lentiviral Transduction for PGRMC1 Knockout
For viral transduction to knockout PGRMC1, ~ 2.5 × 104 NSC34 cells were incubated with the foregoing filtered lentivirus-containing supernatant. After 3 days of transduction, puromycin was added (final 3 μg/ml) to screen sgRNA/Cas9 positive cells. Two weeks later the cell culture was expanded to three 35-mm dishes. In order to assess the efficiency of sgRNA-guided Cas9 cutting in the PGRMC1 genomic sequence (exon-1), genomic DNA was extracted for PCR amplification of the specific region including the sgRNA/Cas9 excision site. Forward primer: gcggaggaagcggactgttc; reverse primer: agcgggccgggggcacgagg. PCR products were digested with 1 μl T7 Endonuclease I (New England Biolabs Inc., MA) for 2 h at room temperature, and then subjected to electrophoresis in a 1.5% agarose gel. PGRMC1 protein knockout was confirmed by Western blotting using a PGRMC1 specific antibody.

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Reviews

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Discussion

Discussion

4 years ago

Author: Mario Udinese Italy

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Discussion

5 years ago

Author: Ben Saar Israel

How to choose a region to target for CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Deletion NSC34 PGRMC1 using lentiCRISPR v2 from Addgene.

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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

Download PDF Download manufacturer protocol

Videos

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