lentiCRISPR v2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.	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.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.
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

Plasmids.
pCDNA 3× FLAG Vpr constructs were described previously (2, 22). The following Vpr sequences were synthesized as Gblocks (IDT) and subcloned into the pCDNA 3× FLAG construct using standard cloning techniques: SE6165, SE9280, ETH2220, SL92B, CFU212, MB897, EK505, Cam13, and CP684. The following consensus Vpr sequences were used: HIV-1 N Vpr (MERAPEDAGPQREPYNEWALELLEELKNEAVRHFPRIWLHGLGQHIYNTYGDTWEGVEAIIRILQQLLFIHYRIGCQHSRIGITPQRRRNGASRS) and HIV-1 O Vpr (MEQAPEDQGPAREPFNEWALELLEELKAEAVRHFPRPWLQALGQYIYETYGDTWVGVMAIIRILQQLLFTHFRIGCQHSRIGINPSNTRGRGRRNGSSRS).

For AAV expression of Vpr, GFP-T2A-3× FLAG Vpr sequences were synthesized (IDT) and cloned into pscAAV-CMV-GFP (48) (generously provided by Dan Stone, Fred Hutchinson Cancer Research Center) by Gibson Assembly (NEB). Lenti-CRISPR V2, psPAX2, and pMD2.G were purchased from Addgene. Guide sequences were synthesized (IDT) and cloned into LentiCRISPR V2 following the Zhang laboratory protocol (49). The guide sequences used were 5′-CACCGTCCTCACAGCCGCTGTTGC-3′ and 5′-AAACGCAACAGCGGCTGTGAGGAC-3′.

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

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Videos

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