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Difficulties with the transfection of cells CHO DG44


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#1 Tellur

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Posted 09 December 2009 - 09:32 AM

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 ((((

#2 Tellur

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Posted 16 December 2009 - 09:28 AM

Hey, people!

Really nobody do not work with these cells?

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

:( :( :(

#3 bob1

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Posted 20 December 2009 - 03:55 PM

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.

#4 Tellur

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Posted 21 December 2009 - 01:42 AM

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.


#5 bob1

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Posted 21 December 2009 - 03:50 PM

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

#6 sara.r

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Posted 21 December 2009 - 10:08 PM

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.

#7 Tellur

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Posted 26 December 2009 - 06:55 AM

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?

#8 sara.r

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Posted 03 January 2010 - 02:25 AM

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.

#9 Tellur

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Posted 10 January 2010 - 11:38 PM

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?

#10 sara.r

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Posted 16 January 2010 - 05:19 AM

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.




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