about the method of transfection - suspection (May/31/2006 )
I have heard a method of transfection, that is, placing the mixed DNA and the reagent to pure cells without adding culture medium or PBS.
Won't the cell be killed by the so high concentration of mix?
Thank you very much for your replies~
-annezhu-
Could u post a link or some website which would describe the transfection. Thanx
-scolix-
Hi annezhu,
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
-Ohmyhead-
QUOTE (annezhu @ May 31 2006, 07:34 AM)
I have heard a method of transfection, that is, placing the mixed DNA and the reagent to pure cells without adding culture medium or PBS.
Won't the cell be killed by the so high concentration of mix?
Thank you very much for your replies~
Won't the cell be killed by the so high concentration of mix?
Thank you very much for your replies~
hello,annezhu,i have seen the method u mentioned in <Molucular Cloning:A Laboratory Manual> ,writen by J.Sambrook E.Fristch T.maniatis .in the book , remove the media , add the mixed DNA to the cells and then incubate the cell under room temperature for 15minutes ,then add new media .(my tongue language is not english ,so i donot know if u can get the meaning )
-chinalilizizi-
QUOTE (Ohmyhead @ Jun 2 2006, 04:43 AM)
Hi annezhu,
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
Do"t you forget to tell that you have to put the mixture in a machine?
I don't know how it works, the provider didn't want to tell, but it seems it's like an electroporation.
-Missele-
QUOTE (Ohmyhead @ Jun 2 2006, 10:43 AM)
Hi annezhu,
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
Thank you for you help, I have read of the Amaxa nucleofector kit optimized for special cell lines, however, I have no idea how to get the kit.
And, is it much more efficient than the lipofectamine?
-annezhu-
QUOTE (chinalilizizi @ Jun 2 2006, 11:43 AM)
hello,annezhu,i have seen the method u mentioned in <Molucular Cloning:A Laboratory Manual> ,writen by J.Sambrook E.Fristch T.maniatis .in the book , remove the media , add the mixed DNA to the cells and then incubate the cell under room temperature for 15minutes ,then add new media .(my tongue language is not english ,so i donot know if u can get the meaning )
Thank you for the help, without your help I would never think of that the Molecular Cloning contains such information of the cell culture.
-annezhu-
QUOTE (Missele @ Jun 2 2006, 04:22 PM)
QUOTE (Ohmyhead @ Jun 2 2006, 04:43 AM)
Hi annezhu,
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
Do"t you forget to tell that you have to put the mixture in a machine?
I don't know how it works, the provider didn't want to tell, but it seems it's like an electroporation.
Do you mean that the Amaxa nucleofector kit just provide a circumstance for electroporation?
-annezhu-
QUOTE (annezhu @ Jun 10 2006, 11:01 AM)
QUOTE (Missele @ Jun 2 2006, 04:22 PM)
QUOTE (Ohmyhead @ Jun 2 2006, 04:43 AM)
Hi annezhu,
I'm doing transfection using Amaxa nucleofector kit. As you described, it's just DNA+reagent+cell, and it works fine. Half of the cells died but most survived cells are transfected. I guess the reagent may contain PBS or some other necessary stuff. But the manufactor did tell me what is the recipe of the reagent.
Do"t you forget to tell that you have to put the mixture in a machine?
I don't know how it works, the provider didn't want to tell, but it seems it's like an electroporation.
Do you mean that the Amaxa nucleofector kit just provide a circumstance for electroporation?
Hi, annezhu:
The story happened in my lab is:
A previous student used lipofectamine to transfect a tough cell line and got 2% transfection effeciency, which was not very helpful. So I was told to use Amaxa and I got 30-50% transfection efficiency. So I think the Amaxa could be much more efficient than the lipofectamine.
To use the Amaxa method, you need both a transfection machine and a kit. The machine might generate a electro pulse so that DNA can be punched into cells (my guess, cause the company didn't tell). The kit provide a magic solution that helps transfection, I have no idea what's in there. See the attachment if you want to know how the machine looks.
As to the question about how to get the kit, I have the fax and tel no. of the Australia distributor. If you are in somewhere else, I think you might want to email their company and ask how to contact a distributor that is best for you.
The company website is: http://www.amaxa.com/
http://www.amaxa.com/contactforms.html
There email is: scientific-support@amaxa.com
Hope this helps!
-Ohmyhead-
I heard that amaxa doesn't work for co-transfection ie if u want to transfect more than 1 plasmid at the same time. Is this true? have u tried it?
-lotusgirl-