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Help, about transfection efficiency


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7 replies to this topic

#1 bluemoon819

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Posted 14 February 2005 - 07:12 AM

Hello everyone:
I am now doing transfection. The cell line I used is NIH3T3 and the plasmid is pEGFP with inserts of 10 kinds of different genes that range from 500bp to 1500bp. I really do not know why the transfection efficiency ranges from 80%~<10%. I did all the transfection parallel and with a control (plasmid without insert) every time. No problems with the control every time.
Anyone have some suggestion? Thank you in advance!

#2 sarah

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Posted 14 February 2005 - 07:39 AM

Hi,

I've done quite a lot of transfections:) and had similar problems:)

How are you propagating your plasmids and how do you check that propagation? Your asnwer could lie in these previous steps.......and the size of your insert can make an impact on your efficiency - do you see the trend that the larger the insert the less efficient your transfection is.


Sarah

#3 badcell

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Posted 14 February 2005 - 09:51 AM

Yeah, I agree with Sarah. I believe the problem is in the plasmid propagation and purification. To minimize this effect I always try to do all the MaxiPreps that I'm going to use on one set of experiments at the same time. And I always dialize the Maxis against TE o/n on the same beaker, to try to get rid of as much salts and contaminats as possible.
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#4 bluemoon819

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Posted 16 February 2005 - 06:38 AM

Thanks sarah and badcell for your suggestion!
Yes, I also think the length of the insert have impact on efficiency. The only one that have 80% transfection efficiency is 500bp and none of the others that long than 800bp have a satifacted efficiency. But why several genes less than 1000 bp have a <40% efficiency? Is about 800bp is difficult for transfection?
We propragation the plasmid in BL21, LB as culture medium.
We extracted and purified the plasmid using QIAGEN Plasmid Maxi Kit (Tip500). Plasmid was eluted using TE and then used directly in transfection without other purification.
What are the best propragation and purification methods?
Thanks again!

Edited by bluemoon819, 16 February 2005 - 06:40 AM.


#5 sarah

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Posted 16 February 2005 - 08:13 AM

hi,

Im not sure what the best procedures are, but I do know that bugs tend to resist propagating plamids with increasing efficacy the larger your insert is (knows this from trying to clone dystrophin!!), jus keep perservering it should work in the end. Everything else sounds okay to me - but have you tried checking the phases of the moon :D

Sarah

#6 bluemoon819

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Posted 17 February 2005 - 06:31 PM

thank sarah very much!
I will try again according to your suggestion.
bluemoon

#7 MaryB

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Posted 19 March 2005 - 07:10 AM

Do any of you know anything about optimising RNA transfection using the GeneJuice transfection reagent? I've only been able to get it owrking with DNA, but never with RNA. Any help would be greatly appreciated!!

#8 joy_maf

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Posted 20 March 2005 - 12:38 PM

Do any of you know anything about optimising RNA transfection using the GeneJuice transfection reagent?  I've only been able to get it owrking with DNA, but never with RNA.  Any help would be greatly appreciated!!

<{POST_SNAPBACK}>



The only reagent we have luck in our lab for both DNA and RNA transfection is GenCarrier-1 on CHO-k1, COS, 3T3, 293 and C2C12 lines. Other brands like Lipofectamin2000, Fugene-6, GenePORTER just couldn't do it.




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