HDR Efficiency: Nucleofection vs. Lipofection

[Karl]

Hi all,

I have read responses here that some investigators have achieved higher HDR efficiency with nucleofection delivery of ssODNs compared to lipofection.  I don't have access to the Amaxa system but do have access to the BioRad Gene Pulser Xcell Electroporation System.  Has anyone used this with any success?  Particularly curious about parameters including DNA amounts, cell number used and "electroporation buffer".  I have the PX459 vector and have followed the 500ng PX459 w/ 1uL of ssODN (10uM) lipofectamine transfection protocol but receive a very low transfection efficiency (measured as 0 colonies returned after puromycin selection @ 3 ug/mL in 293FT cells).  The PX459 alone transfection returns successfully transfected, puromycin-resistant cells in large numbers so I am searching for a way to achieve a more efficient delivery of both nucleic acids.  Any and all comments are greatly appreciated.

Cheers,

Karl

[Vladimir Gainullin]

It should be exactly the same thing, you will have to determine transfection conditions empirically for your cell line. You can find a transfection protocol for a similar cell type and start from there. Make sure you get the right cuvette gap for mammalian cells.

Here is the electroporation buffer that gives consistently high transfection efficiency 

Electroporation Solution

 

20 mM HEPES

135 mM KCl

2 mM MgCl2

0.5% Ficoll 400,

pH adjusted to 7.6 with NaOH.

filter-sterlilize

 

ATP/Glutathione

Add 1/100 of stock ATP/Glut to cell right before or right after electroporation (only needed if cells tend to die after electroporation)

Stock

200 mM ATP

500 mM glutathione.

pH – 7.0

[Duško Lainšček]

dear all,

i really need help,because i do not know how to calculate integrated intensity using Image J,because i am geting very strange numbers of HR...

in need of help.

best

dusko

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[Ran Antes]

Hi all,

I have a similar but different question:

I am trying to transfect my mESC and I am using the amaxa nuleofector.

My problem is that the efficiency is really low, most of the cells die and only few express GFP (using the px461).

I know there are 4 different programs on this machine for mESC. Did anyone use it? can you recommend a specific program?

Thanks,

Ran

[Xue Mei]

Hi all,

I have a similar question here as well. I was wondering what would be a good program for EB3/5 cells on the Gene Pulser.  I tried the Gene Pulser Xcell once, 25ug DNA, 2million cells, 240volts, 500uF and selected with 0.5ug/mL pyro, cells did not die but T7E1 showed that there is no NHEJ at all. I am in the process of doing a puro dose curve and at the same time I plan to tweak the program and try an empty px458 next time. 

Any suggestions will be greatly appreciated.

Xue

On Wednesday, January 7, 2015 at 9:21:49 AM UTC-5, Karl wrote:

[Xue Mei]

Karl,

Have you had any success with Gene Pulser so far? 

Thanks,