pLKO5.sgRNA.EFS.GFP

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

Generation of stable MC3T3-e1 clonal cell lines
Lentivirus production: HEK293T cells plated in 10-cm dishes seeded at ∼80% confluency were transfected with 12 μg lentiviral dCas9 fusion or gRNA constructs, 6 μg of the helper plasmids VSVG and Δ8.9 each, and Lipofectamine 2000 according to the manufacturer's instruction. Viral supernatant was collected 48 h post transfection, filtered with a 0.45 μM polyvinylidene difluoride filter (Millipore), aliquoted and stored at −80 °C.

Lentiviral transduction: MC3T3-e1 cells were transduced first with dCas9 fusion lentivirus via spinfection in six-well plates containing 2 ml of lentiviral supernatant media supplemented with 10 μg ml−1 polybrene (Santa Cruz, cat. #sc-134220). Plates were centrifuged for 30 min at 1,000g, 37 °C. Media was immediately replaced with fresh alpha minimum essential media. Cells were propagated for 1 week before another round of infection with gRNA-3 lentivirus in the same manner as described above. Lentivirus-infected cells were then singly sorted according to GFP by FACS into 96-well plates using the BD FACS Aria II and expanded to generate clonal cell lines.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pLKO5.sgRNA.EFS.GFP below.

Download manufacturer protocol

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