Articles, announcements and insights from Horizon Discovery

[Protocol] How to bank cell lines

Jun 10, 2016 1:26:45 PM No Comments

Whether it's a stock you've expanded following it's arrival from the cell bank, or a clone you've carefully nurtured from the single cell, banking down your cells in the right way is crucial if you're going to be able to return to them time and again, and revive them quickly so that you can get on with your experiments.

Here's our protocol for banking human cell lines:

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[Protocol] Optimizing transfection for genome editing

Jun 10, 2016 1:25:54 PM No Comments

Transfection or electroporation is used to efficiently introduce the nucleic acids required for CRISPR cell line engineering into a cell line. The type and number of nucleic acids (usually plasmids) being introduced into a cell line will depend on the engineering event being undertaken. At its most simple, gene knockouts can be achieved by transfection of a plasmid expressing wild-type Cas9 along with a guide RNA (gRNA) to the gene that is being knocked out.

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[Protocol] Harvesting and analyzing clones following CRISPR gene editing

Jun 10, 2016 1:25:18 PM No Comments

If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions, and have a good idea of your guide's editing efficiency. This number should give you a rough idea of how many clones you're going to have to screen to find a targeted clones. Keep in mind the following:

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[Protocol] Expansion of cell line clones from single cells

Jun 10, 2016 1:23:34 PM No Comments

Once a clone has been identified positive for targeting in the initial screen, the cells should be expanded to a 48-well plate. Expansion of the cells can usually occur at 1-4 days post screen.

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A single cell dilution protocol for obtaining CRISPR targeted cell clones

Jun 8, 2016 3:57:22 PM No Comments

Following transfection of CRISPR reagents, cells will need to be single cell diluted to obtain a clonal population. There are several ways of doing this. Below we detail the single cell dilution protocol used at Horizon.

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If a cell line tolerates being single cell diluted then plating 96-well plates at 1 cell per well in standard cell culture media is appropriate.

If the cell line does not tolerate single cell dilution in standard media, then the use of conditioned media can often improve clone recovery. If the use of conditioned media in 96 well plates does not improve the recovery of clones, cells can be plated to large tissue culture dishes and individual colonies picked.

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