Production of LentiCRISPR Viruses: Difference between revisions

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* Thaw and passage cells. Use passage <20. These amounts are for one plasmid.

* Split cells normally each time seeding 7 x 105 cells in a 10 cm dish, in DMEM/PSG/10% FBS.

* The day before the transfection, seed 3.8 x 106 cells into a fresh plate. Seed cells into media '''without PSG'''

* The day before the transfection, seed 3.8 x 106 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"

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* Add transfection mixture slowly to the cells.

* 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.

* Centrifuge at 500g for 5 minutes to pellet any cells.

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[[ Category: Transfection ]]

[[ Category: Molecular Biology ]]

__NOTOC__

@@ Line 12: / Line 12: @@
 * 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'''
+* 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"
@@ Line 47: / Line 50: @@
 * Add transfection mixture slowly to the cells.
 * 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.
 * Centrifuge at 500g for 5 minutes to pellet any cells.
@@ Line 56: / Line 61: @@
 [[ Category: Transfection ]]
 [[ Category: Molecular Biology ]]
+__NOTOC__