Login

Join for Free!
118318 members


AMP selection of HEK293

Discussion of all aspects of cellular structure, physiology and communication.

Moderator: BioTeam

AMP selection of HEK293

Postby szkornelia » Wed Jul 19, 2006 12:35 pm

Hello,

I am transfecting HEK293 cells, and trying to make a stable cell line containing an Amp resistant plasmid construct. Does anyone can tell me the concentration of the Amp I should use when I am trying to select the plasmid containing cells? Any help would be appreciated.

Thanks

Kornelia
szkornelia
Garter
Garter
 
Posts: 2
Joined: Wed Jul 19, 2006 12:31 pm

Postby TerAle » Wed Jul 19, 2006 7:11 pm

Dear Kornelia,

The "Amp", if I understood right, is the ampicillin resistance gene. Ampicillin is the prokariotic antibiotic and is used for selection of bacteria. To select transfected eukariotic cells (HEK293 or any other) your plasmid should have a gene providing the resistance to an eukariotic antibiotic, which is in your case, most probably, the Neo (neomicin) resistancy gene. Check your plasmid for gene like this. If there is no genes for eukariotic antibiotic resistance then you have to use another plasmid.

Good luck!

Alexei.
TerAle
Garter
Garter
 
Posts: 2
Joined: Wed Jul 19, 2006 7:02 pm

Postby TerAle » Wed Jul 19, 2006 7:14 pm

Sure, it should be "neomycin".

Sorry.

Alexei
TerAle
Garter
Garter
 
Posts: 2
Joined: Wed Jul 19, 2006 7:02 pm


Postby damien james » Thu Jul 20, 2006 1:54 am

@TerAle: He probably knows all that. That not telling nothing, just basic micro. He just want concentration of Amp.

You should use multiple Amp dilution instead of just one single one and plate multiple times with different dilution factor. Well, you know. I would start with 250-300 mg per mL. But I would not dilute by 10s because that is too much. Maybe 1/2s or less. To like 150, 100, you get idea.
The hand of God may well be all around us, but it is not, nor can it be, the task of science to dust for fingerprints.
User avatar
damien james
Coral
Coral
 
Posts: 237
Joined: Thu Mar 02, 2006 5:51 am
Location: U.S.

Postby canalon » Thu Jul 20, 2006 1:59 am

Well most of the plasmid I have used were selected with 100 mg/L or ug/mL, not 200 or 300 mg/ml. That would be expensive too ;)
But the way the question is asked I think that TerAle makes a more interesting point.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
User avatar
canalon
Inland Taipan
Inland Taipan
 
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Postby damien james » Thu Jul 20, 2006 2:21 am

I thought szkornelia was only asking for concentration of Amp, since that was his question. If you are selecting for Amp resistant HEK293 line and do not know already micro, then you should not be messing with it in first place.

Also you are right, it should be ug/mL. I made mistake in post. Do not know what I was thinking. I was thinking about how we buy Amp instead of dilution with plate or broth. Do you use trihydrate form? We buy in several different dilution. 250mg/ml, then other are about half that.
The hand of God may well be all around us, but it is not, nor can it be, the task of science to dust for fingerprints.
User avatar
damien james
Coral
Coral
 
Posts: 237
Joined: Thu Mar 02, 2006 5:51 am
Location: U.S.

Postby szkornelia » Thu Jul 20, 2006 6:27 am

Thanks for everybody. I meant Neo, I don't know why did I write Amp. Maybe because I was picking colonies from the transformed bacterial plate today, and the procaryotic resistancy gene in the construct is Amp. Thanks again for the suggestions.
szkornelia
Garter
Garter
 
Posts: 2
Joined: Wed Jul 19, 2006 12:31 pm


Return to Cell Biology

Who is online

Users browsing this forum: No registered users and 4 guests