Production of LentiCRISPR Viruses - Revision history

Davebrid: Removed table of contents

2017-12-08T16:20:28Z

Removed table of contents

← Older revision		Revision as of 16:20, 8 December 2017
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	[[ Category: Transfection ]]		[[ Category: Transfection ]]
	[[ Category: Molecular Biology ]]		[[ Category: Molecular Biology ]]
			__NOTOC__

Davebrid: Made new sections and added part about chloroquine

2017-12-08T16:19:43Z

Made new sections and added part about chloroquine

← Older revision		Revision as of 16:19, 8 December 2017
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	* Thaw and passage cells. Use passage <20. These amounts are for one plasmid.		* Thaw and passage cells. Use passage <20. These amounts are for one plasmid.
	* Split cells normally each time seeding 7 x 10<sup>5</sup> cells in a 10 cm dish, in DMEM/PSG/10% FBS.		* Split cells normally each time seeding 7 x 10<sup>5</sup> cells in a 10 cm dish, in DMEM/PSG/10% FBS.
	* The day before the transfection, seed 3.8 x 10<sup>6</sup> cells into a fresh plate. Seed cells into media '''without PSG'''		* The day before the transfection, seed 3.8 x 10<sup>6</sup> cells into a fresh plate. Seed cells into media '''without PSG'''.
	* Prepare DNA according to this table in a sterile 2 mL tube.:
			==Transfection Procedure==
			* The morning of the transfection day, replace the media with fresh DMEM '''without PSG''' and containing 10 uL of 25 mM chloroquine. Wait ~5h before going onto the next step.
			* Prepare DNA according to this table in a sterile 2 mL tube:

	{\| class="wikitable"		{\| class="wikitable"
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	* Add transfection mixture slowly to the cells.		* Add transfection mixture slowly to the cells.
	* Incubate overnight. The next day carefully replace with media containing PSG.		* Incubate overnight. The next day carefully replace with media containing PSG.

			==Collecting Viral Particles==
	* Collect media at 96h, or at 48, 72 and 96h post infection. Combine supernatants in a 50 mL conical tube.		* Collect media at 96h, or at 48, 72 and 96h post infection. Combine supernatants in a 50 mL conical tube.
	* Centrifuge at 500g for 5 minutes to pellet any cells.		* Centrifuge at 500g for 5 minutes to pellet any cells.

Davebrid: Added categories

2017-12-08T16:13:27Z

Added categories

← Older revision		Revision as of 16:13, 8 December 2017
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	* Centrifuge at 500g for 5 minutes to pellet any cells.		* Centrifuge at 500g for 5 minutes to pellet any cells.
	* Filter supernatant through the 0.45 um. Aliquot and snap freeze in liquid nitrogen in screw capped vials.		* Filter supernatant through the 0.45 um. Aliquot and snap freeze in liquid nitrogen in screw capped vials.

			[[ Category: CRISPR ]]
			[[ Category: Cell Culture ]]
			[[ Category: Tissue Culture ]]
			[[ Category: Transfection ]]
			[[ Category: Molecular Biology ]]

Davebrid: Wrote initial protocol for virus production.

2017-12-08T16:12:59Z

Wrote initial protocol for virus production.

New page

This protocol is modified from the [https://www.addgene.org/protocols/lentivirus-production/ Addgene website] protocol

== Materials==
* Use the 293T/17 cells (purchased as a stock from ATCC and aliquoted in separate box in the liquid nitrogen; [https://www.atcc.org/Products/All/CRL-11268.aspx ATCC page]).
* Plasmids (lenti-vector, psPAX2, pMD2.G)
* OptiMEM
* Chloroquine stocks (25 mM stocks). See [[Preparation of Chloroquine Stocks]].
* PEI (1 mg/mL stocks). See [[Preparation of PEI Stocks]].
* DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG

==Preparation of Cells==
* Thaw and passage cells. Use passage <20. These amounts are for one plasmid.
* Split cells normally each time seeding 7 x 10<sup>5</sup> cells in a 10 cm dish, in DMEM/PSG/10% FBS.
* The day before the transfection, seed 3.8 x 10<sup>6</sup> cells into a fresh plate. Seed cells into media '''without PSG'''
* Prepare DNA according to this table in a sterile 2 mL tube.:

{| class="wikitable"
! style="text-align: center; font-weight: bold;" | Reagent
! style="font-weight: bold;" | pmoles
! style="font-weight: bold;" | ug
! style="font-weight: bold;" | Volume Added
|-
| style="text-align: center;" | psPAX2
| 1.3
| 9.2
|
|-
| style="text-align: center;" | pMD2.G
| 0.72
| 2.77
|
|-
| style="text-align: center;" | LentiCRISPRv2-sgRNA
| 1.64
| 16.1
|
|-
| style="text-align: center;" | OptiMEM
| -
| -
| 500 uL
|}

* For this amount of DNA (28 ug) try using 3:1 PEI:DNA, so add 84 uL PEI into 1 mL OptiMEM.
* Gently add the PEI solution dropwise into the DNA solution.
* Incubate at room temperature for 15-20min.
* Add transfection mixture slowly to the cells.
* Incubate overnight. The next day carefully replace with media containing PSG.
* Collect media at 96h, or at 48, 72 and 96h post infection. Combine supernatants in a 50 mL conical tube.
* Centrifuge at 500g for 5 minutes to pellet any cells.
* Filter supernatant through the 0.45 um. Aliquot and snap freeze in liquid nitrogen in screw capped vials.