9 replies to this topic

[Tellur]

We purchased OptiCHO ™ Antibody Express Kit. But the cells do not transfect with original transfectagent. They can not be transfected and with the help with other Lipoftagents (Lipofectamine and other). Usually we have only 0.3 - 5 percent effency of transfection. We increase the amount of FreeStyle MAX reaget, we have mass cell death during transfection (and 5% percent of tansfectants), but the cells which stay alive do not duplicate and die during selection.
CaPi transfection method do not work anyway.
I'm confused ((((

[Tellur]

Hey, people!

Really nobody do not work with these cells?

It's a big trouble for me to transfect these cells and select them...

[bob1]

Talk to the supplier, they can usually offer some help.  It may be that the kit is bad, and they can send you another one.

[Tellur]

Thank you for your reply.
Supplier of kit is Invitrogen. We talked with them. They said that they have 60% of transfected cells. Nothing did not want to send.

You worked with these cells?

Forgive me for my bad English

Edited by Tellur, 21 December 2009 - 01:43 AM.

[bob1]

Sorry, no I don't work with these cells or the kit; I was just offering general advice.

[sara.r]

Hi
We have successfull transfection (up to 50 percent at least) of DG44 suspention cells in CDG44 medium with lipofectamin 2000 CD I think lipofectamin 2000 might help too.

[Tellur]

sara.r, on Dec 22 2009, 09:08 AM, said:

Hi
We have successfull transfection (up to 50 percent at least) of DG44 suspention cells in CDG44 medium with lipofectamin 2000 CD I think lipofectamin 2000 might help too.

Thank you for reply.
Which method you used for purification of DNA. How much DNA you use? What ratio of DNA/Lipofectamine you used? You used a circular or linear DNA?

[sara.r]

Tellur, on Dec 26 2009, 06:55 AM, said:

sara.r, on Dec 22 2009, 09:08 AM, said:

Hi
We have successfull transfection (up to 50 percent at least) of DG44 suspention cells in CDG44 medium with lipofectamin 2000 CD I think lipofectamin 2000 might help too.

Thank you for reply.
Which method you used for purification of DNA. How much DNA you use? What ratio of DNA/Lipofectamine you used? You used a circular or linear DNA?

sorry for too late reply
we use reqular miniprep plasmid extraction kit for plasmid purification as I can remember we use about 3 microgram plasmid with 10 microliter lipofectamin 2000 cd, there is a protocol in dg44 kit for this that mentioned the exact ratio.

[Tellur]

To sara.r

Thans a lot.
What yield of protein you harvest from liter/milliliter of culture? Have you used serum free media or media with serum?

[sara.r]

Tellur, on Jan 11 2010, 12:38 AM, said:

To sara.r

Thans a lot.
What yield of protein you harvest from liter/milliliter of culture? Have you used serum free media or media with serum?

hi
I only was involved to transfection optimization by gfp expressing vector, but as I remember my collegues get 1-3 pg/ml using suspend dg44 in  serum free  cd  dg44 medium without dhfr amplification.