Log In Sign up
The largest database of trusted experimental protocols

Pklv2.2 h7skgrna5 sapi hu6grna5 bbsi pgkpurobfp w

Manufactured by Addgene
Visit Supplier

PKLV2.2-h7SKgRNA5(SapI)-hU6gRNA5(BbsI)-PGKpuroBFP-W is a plasmid vector that can be used for gene editing applications. It contains two guide RNA expression cassettes, a puromycin resistance gene, and a blue fluorescent protein (BFP) reporter.

Automatically generated - may contain errors

Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Ta cloning, by Promega (2 mentions) X vivo medium, by Lonza (2 mentions) Pl crispr, by Addgene (1 mentions) Lenticrispr v2, by Addgene (1 mentions)

3 protocols using pklv2.2 h7skgrna5 sapi hu6grna5 bbsi pgkpurobfp w

1

Single gRNA CRISPR Plasmid Cloning

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Single gRNAs targeting the miRNA regions were cloned into LentiCRISPR V2 (a gift from Feng Zhang, Addgene plasmid #52961) or pKLV2.2-h7SKgRNA5(SapI)-hU6gRNA5(BbsI)-PGKpuroBFP-W (Addgene plasmid # 72666) or pKLV2.2-mU6gRNA5(SapI)-hU6gRNA5(BbsI)-PGKpuroBFP-W (Addgene no#72666) (gifts from Kosuke Yusa) (Tzelepis et al., 2016 (link); Ihry et al., 2018 (link)). The gRNAs were cloned at the BsmBI site for LentiCRISPR V2 plasmid and SapI or BbsI sites for the pKLV2.2 plasmids as previously described (Sanjana et al., 2014 (link); Tzelepis et al., 2016 (link)). Single gRNAs targeting the γ globin promoter region were cloned into pL.CRISPR.EFS.GFP (Addgene plasmid #57818) (gift from Benjamin Ebert) at the BsmBI site (Heckl et al., 2014 (link)).
Ijee S., Chambayil K., Chaudhury A.D., Bagchi A., Modak K., Das S., Benjamin E.S., Rani S., Paul D.Z., Nath A., Roy D., Palani D., Priyanka S., Ravichandran R., Kumary B.K., Sivamani Y., S. V., Babu D., Nakamura Y., Thamodaran V., Balasubramanian P, & Velayudhan S.R. (2024). Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs. Frontiers in Molecular Biosciences, 10, 1295507.
+ Open protocol
+ Expand
2

CRISPR-mediated Deletion of Regulatory Elements in Mouse Leukemia Cells

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Mouse leukemia cells carrying Npm1c/Flt3-ITD/Cas9 (termed DM-Cas9 cells)42 (link) were cultured in X-Vivo medium (Lonza) plus 10% fetal bovine serum (Gibco) supplemented with cytokines as described above. In the presence of Cas9, using dual guide RNAs (gRNAs) to specifically target two genomic loci has been demonstrated previously52 (link). Paired dual gRNAs targeting specific cis-regulatory elements or scramble control dual gRNAs were designed with IDT web tools (https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM) and their sequences were available in Supplementary Table 12. Oligos of dual gRNAs were cloned into lentivirual vector pKLV2.2-h7SKgRNA5(SapI)-hU6gRNA5(BbsI)-PGKpuroBFP-W (#72666, Addgene). Production of gRNA particles and transduction of DM-Cas9 cells were carried out same as for shRNA. To confirm the target deletion in the bulk transduced cells, PCR primers were designed to amplify the wildtype or modified allele (Supplementary Table 12). Size of PCR products was checked by agarose gel electrophoresis. Furthermore, PCR products were subject to TA cloning (Promega). Plasmid DNA was extracted from individual colonies and was confirmed for deletion by Sanger sequencing.
Yun H., Narayan N., Vohra S., Giotopoulos G., Mupo A., Madrigal P., Sasca D., Lara-Astiaso D., Horton S.J., Agrawal-Singh S., Meduri E., Basheer F., Marando L., Gozdecka M., Dovey O.M., Castillo-Venzor A., Wang X., Gallipoli P., Müller-Tidow C., Osborne C.S., Vassiliou G.S, & Huntly B.J. (2021). Mutational synergy during leukemia induction remodels chromatin accessibility, modification and 3-Dimensional DNA topology to alter gene expression. Nature genetics, 53(10), 1443-1455.
+ Open protocol
+ Expand
3

CRISPR-mediated Deletion of Regulatory Elements in Mouse Leukemia Cells

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Mouse leukemia cells carrying Npm1c/Flt3-ITD/Cas9 (termed DM-Cas9 cells)42 (link) were cultured in X-Vivo medium (Lonza) plus 10% fetal bovine serum (Gibco) supplemented with cytokines as described above. In the presence of Cas9, using dual guide RNAs (gRNAs) to specifically target two genomic loci has been demonstrated previously52 (link). Paired dual gRNAs targeting specific cis-regulatory elements or scramble control dual gRNAs were designed with IDT web tools (https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM) and their sequences were available in Supplementary Table 12. Oligos of dual gRNAs were cloned into lentivirual vector pKLV2.2-h7SKgRNA5(SapI)-hU6gRNA5(BbsI)-PGKpuroBFP-W (#72666, Addgene). Production of gRNA particles and transduction of DM-Cas9 cells were carried out same as for shRNA. To confirm the target deletion in the bulk transduced cells, PCR primers were designed to amplify the wildtype or modified allele (Supplementary Table 12). Size of PCR products was checked by agarose gel electrophoresis. Furthermore, PCR products were subject to TA cloning (Promega). Plasmid DNA was extracted from individual colonies and was confirmed for deletion by Sanger sequencing.
Yun H., Narayan N., Vohra S., Giotopoulos G., Mupo A., Madrigal P., Sasca D., Lara-Astiaso D., Horton S.J., Agrawal-Singh S., Meduri E., Basheer F., Marando L., Gozdecka M., Dovey O.M., Castillo-Venzor A., Wang X., Gallipoli P., Müller-Tidow C., Osborne C.S., Vassiliou G.S, & Huntly B.J. (2021). Mutational synergy during leukemia induction remodels chromatin accessibility, modification and 3-Dimensional DNA topology to alter gene expression. Nature genetics, 53(10), 1443-1455.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!