lentiCas9-Blast

Protocol tips

Upstream tips	Protocol tips	Downstream 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.

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

Knockout cell lines
Constructs were packaged into lentivirus in HEK 293T cells using the Fugene HD transfection reagent (Promega) and 2 μg of the vector, 2 μg psPAX2, and 0.2 μg pHEF-VSV-G. THP-1 or RAW 264.7 cells were spinfected with virus for 2 h at 1000g at 30 °C supplemented 8 μg/mL polybrene. After 2 days, cells were selected for stable expression of S. pyogenes Cas9 (Addgene #52962) using blasticidin (5 μg/mL for THP-1, 1 μg/mL for RAW 264.7) and for stable expression of sgRNAs using puromycin (0.5 μg/mL for THP-1, 5 μg/mL for RAW 264.7) or hygromycin (100 μg/mL for THP-1). After 10 d, the cells were harvested for immunoblotting or experiments. To confirm allelic modifications, genomic DNA was harvested using Blood and Tissue Culture Kit (Qiagen). A 600 base pair region containing the sgRNA cut site was then PCR amplified and subcloned using the CloneJET PCR Cloning Kit (ThermoFisher). Single bacterial colonies were isolated and sequenced.



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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for lentiCas9-Blast below.

Download manufacturer protocol

Videos

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