lenti dCAS-VP64_Blast

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Protocol tips
The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

Publication protocol

Plasmid and Vector Constructs
Plasmids encoding NLS-dCas9-VP64 and MS2-p65-HSF1
(plasmid #61425 and #61426), and dCas9-24xGCN4-v4 and scFvGCN4-sfGFP-VP64 expression plasmids were obtained from
Addgene (plasmid #60910 and #60904). The sgRNA expression
plasmid was obtained from Addgene (plasmid #61427), and the
multiple sgRNA clone vector including BsmBI restriction sites
facilitated the insertion of different gRNA target corresponding
oligo DNAs. The lentiCRISPR-V2 plasmid was obtained from
Addgene (plasmid #52961) and the pGL3-basic plasmid was
obtained from Promega. To obtain the interferon-γ promoter
luciferase reporter plasmid, the promoter fragment was amplified
from peripheral blood mononuclear cells using the forward primer
F (5’-CGGGGTACCGGGATTACAGGCGTGAGCCACC-3’) and the
reverse primer R (5’-CCGCTCGAGCGTAGATATTGCAGATAC
TTCTGGGCTT-3’), followed by purification and digestion by KpnI
and XhoI restriction enzymes and then ligation into the KpnI-XhoI
clone sites of the pGL3-basic plasmid. Interferon-γ promoter
reporter lacking the sgRNA target site (ΔsgRNA9 and sgRNA10)-
luc was constructed using two-step fusion PCR: forward primer
(5’-CGGGGTACCGGGATTACAGGCGTGAGCCACC-3’) and reverse
primer (5’-CGTATTTTCACAAGTTTTTTAATGATAGTTTGTAT-3’)
amplify upstream of the 5’-promoter; forward primer (5’-TTA
TTAATACAAACTATCATTAAAAAACTTGTG-3’) and reverse
primer (5’-CCGCTCGAGCGTAGATATTGCAGATACTTCTGGGCTT3’) amplify downstream of the 5’-promoter. Finally, a full-length
interferon-γ lacking the sgRNA9 and sgRNA10 target site fragments
was amplified using a forward primer (5’-CGGGGTACCGGG
ATTACAGGCGTGAGCCACC-3’) and a reverse primer (5’-CCG
CTCGAGCGTAGATATTGCAGATACTTCTGGGCTT-3’) with two
fragments amplified in the first step as templates. The purified
interferon-γ promoter (ΔsgRNA9 and sgRNA10) PCR product was
then ligated into the KpnI- and XhoI-digested pGL3-basic plasmid.
All the expression plasmids were confirmed by sequencing

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Papers

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Manufacturer protocol

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Download manufacturer protocol

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