Troubleshooting for transfection of the CRISPR/Cas9 using lipofectamine

[Alaa Fisal]

what are the best conditions to transfect the cell line with CRISPR/Cas9 using lipofectamine reagent? I used lipofectamine 2000 reagent for transfection but the efficiency were very low

[Michael Goodman]

I was recently having issues using lipofectamine 3000 so did a lot of troubleshooting.  There may be quite a few cell specific issues when it comes to this topic, in particular if your cells are adherent or suspension.  What cell line are you using.  Also, I would advise using less cells than what the manual suggests.  What variables have you tried adjusting, e.g. amount of lipofectamine?  Are you transfecting with DNA or RNA?

Michael

[tash...@gmail.com]

It depends on your cell line - HEK293 and HeLa etc. work very well but most others just don't respond well to lipofectamine. I have used both lipofectamine 2000 and 3000 and always get the same results; you need to flow sort or do puromycin selection to enrich for your transfected cells.

[Alaa Fisal]

Actually I am using T-47,D and I am transfecting it with CRISPR/Cas9 plasmid and GFP plasmid as a positive control but nearly no transfection occurred, and unfortunately the plasmid has no selection marker. I am seeking that if any one has an advice about the quantity plasmids or the lipofectamine 2000 itself I mean any modification that could help.  

[Hind M Naffadi]

Dear Alaa

I have the same problem now I am trying to transfect my cell MDA-MB-231 with CRISPR/CAS9 plasmid with lipofectamine 3000, every time I got low transfection efficiency about 10%. So, Could you tell me if your transfection work and dose your CRISPR work?

[Anoeska]

It can be smart to test some other transfection reagents. Try to contact companies selling them and tell them about your problem.

They often want you to try (and later buy) their products so make use of that (ask for a free sample to try for example).

Or go for electroporation if you have a machine available.

If you cannot switch, try enriching for transfected cells for example via FACS or puro-selection.

Anoeska

On Friday, April 17, 2015 at 7:15:09 PM UTC+2, Alaa Fisal wrote:

[Razieh Monjezi]

Hi Michae, 

do you have any experience with suspension Jurkat cells transfection with mRNA? which transfection reagent do you suggest for suspension cells? what is the difference between suspesion and adherent cells for transfection? 

regards 

Razieh

[Matthew Thompson]

The best conditions vary by cell type, as does Lipofectamine's effectiveness. They show on their website that some cell types are unable to break 30% transfection efficiency. It could also be the plasmid that has a problem - if it is not pure enough or at the right concentration at transfection. If you haven't found any solid results in the literature for good conditions using your specific reagents and cell type, it would be good to do a transfection optimization experiment before switching reagents and running into the same problem. The variables to test include the cell seed density, plasmid amount, and Lipofectamine reagent amount, using the Lipofectamine protocol as a guide for selecting values for these parameters.

On Friday, April 17, 2015 at 1:15:09 PM UTC-4, Alaa Fisal wrote: