Glioblastoma cell lines are kind of hard to transfect. Here are a few pointers that really affect the efficicency:

Make sure the DNA quality is good (midi prep at least- and endotoxin free!) this is why increased plasmid is toxic to cells in general.

If you have standardize say 1:5 or 1:10 ratio of DNA to ultracruze make sure you upscale it accordingly by say using 3ug of DNA and 15ul of ultracruz. 

One of the most important problem with these cells is the seeding density. What density do you seed your cells at? This is very important to be standardized. If your cells are at low confluency then the same reagent you previously tested will be toxic to small number of cells. Also if its slightly higher than standardized conditions the cells will not get transfected. (Similar to what you are observing) My cells act similarly at give me the best transfection at 65-70%. Anything above or below doesn't work. So make sure you test this as well when you are scaling up your reagents.

I hope this helps :)

are you using the sgguide /cas9 plus and HDR plasmid?

i don't like the SC  systems ..the vectors you purchase cannot be amplify easely (but I bet they are using the RNA-out ecoli strain to manufacture the vectors) .

but basically, if your guide is effective. i suggest to do what i do with pretty good success .

- co transfect your cells with your guide/cas9 vectors (it encode a gfp also) to the transfection mix add 1/20 (molecul/molecules) of for exmple an empty puromycine vector

- 24h post transfection, apply puromycine  (you should have performed a kill curve to know the concentration able to kill 100% non transfected cells.

-48h later the ctl cells have probably died.

- seed the cells for colony formation (adherent cells) or clone your cells with facs sorting (you can keep the cells as a pool and apply any test you want pcr, wb, etc )

- amplify the clones and proceed to clone validation...by pcr /wb/if as necessary

this work nicely

hope this help

didier

Glioblastoma cell lines are kind of hard to transfect. Here are a few pointers that really affect the efficicency:

Make sure the DNA quality is good (midi prep at least- and endotoxin free!) this is why increased plasmid is toxic to cells in general.

If you have standardize say 1:5 or 1:10 ratio of DNA to ultracruze make sure you upscale it accordingly by say using 3ug of DNA and 15ul of ultracruz. 

One of the most important problem with these cells is the seeding density. What density do you seed your cells at? This is very important to be standardized. If your cells are at low confluency then the same reagent you previously tested will be toxic to small number of cells. Also if its slightly higher than standardized conditions the cells will not get transfected. (Similar to what you are observing) My cells act similarly at give me the best transfection at 65-70%. Anything above or below doesn't work. So make sure you test this as well when you are scaling up your reagents.

I hope this helps :)

Thank you. I've tried everything, i had promising result at 24 hrs but by 48 hrs I observed very unhealthy cells. We will be moving on to another method of KO.

Hi! In our hands the idea of  Magnetofection worked very well from hard to kill/hard to transfect to easy to kill/hard to transfect cells. they have different products for different cell types. after the Lipofectamine2000 was finished (from the kit) we started using homemade PEI with those magnetic particles- that makes transfection as efficient and cheap (even HUVEC cell line had really nice transfection rate). one doesn;t need to increase toxicity with higher DNA:lipofectant ratio. here's some info about the Magnetofection:

http://www.ozbiosciences.com/content/13-magnetofection-transfection