lentiGuide-Puro

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

Hit validation by CRISPR/Cas9-mediated gene knockout
Cas9-expressing HEK-293T and A549 cells were generated by transducing them first with lentiviruses based on the plasmid lentiCas9-Blast (Addgene number 52962), followed by selection with blasticidine for 10 days. Cells were then transduced with lentiviruses derived from the plasmid lentiGuide-Puro44 (Addgene number 52963), which leads to the expression of specific gRNAs) listed in Supplementary Data 4. Cas9-positive HEK-293T cells expressing individual gRNAs were generated by keeping cells under selection medium containing blasticidine (5 μg ml−1) and puromycin (2 μg ml−1) for an additional 10 days. A549 cells depleted for CLK1 were generated accordingly, but in this case single-cell clones were generated, which were kept under selection medium containing blasticidine (10 μg ml−1) and puromycin (2.5 μg ml−1) for an additional 10 days and subsequently analysed using the GeneArt Genomic Cleavage Detection Kit (Life Technologies) and the following oligonucleotides: CLK1_up: 5′-TGGAGTAGAGTGGCACGATG-3′; CLK1_down: 5′-GGATGCTTTAAGGCTTCTCTGA-3′ (uncropped gel presented in Supplementary Fig. 6). After the selection process, gRNA-expressing HEK-293T and A549 cells were infected with CHIKV-GFP for the periods of time and at MOIs as indicated.



Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Deletion CLK1 using lentiGuide-Puro from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for lentiGuide-Puro below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Human - Deletion CLK1 using lentiGuide-Puro from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.