On 5 nov. 09, at 00:37, Deen wrote:

> Liao, Xin-hua (NIH/NIDDK) [F] wrote:
>> Hi, dear all,
>> Since I joined Dicty community, I encountered a lot of technique  
>> problems or confusions. Some of them may be not a real problem, and  
>> the question may be stupid, but they all bother me for a long time.  
>> I put them all here, hope you can help me clarify them.
>>
>> 1.      Blasticidin selection: for knock out, after transformation  
>> for around 5 days under blasticidin selection, I transfer single  
>> colonies under medium to 96-well plate. For 96 colonies, only a few  
>> of them (usually around 10) can really grow up in the wells again.  
>> It looks to me a lot of "fake" colonies survive through the drug  
>> selection. If the recombination rate happens to be low, this  
>> phenomenon will make the screening very difficult to me. 5ug/ml of  
>> blasticidin is used. (This concentration is enough to stop WT cells  
>> growing and kill them under shaking condition, though).
>>
> this is based on my experience some ax2 cells are slightly resistant  
> to Bsr even when we used at 5ug/ml Bsr we got some fake colonies  
> (but not 90% as you see) but to avoid this we do selection at 10ug/ 
> ml Bsr. Sometimes longer selection in Bsr is also a solution (may be  
> 2 weeks). Based on the number of colonies on your plate you can  
> decide whether to gradually increase the drug concentration or  
> select for longer or may be combination of both may help resolve  
> your problem.
>
>> 2.      G418 selection: for overexpression, after transformation  
>> for around 5 days under G418 selection, I spread single cell on KA  
>> plate, and then pick single colony to culture in 12-well plate. A  
>> lot of them can not expand in the well anymore. Again, I suspect  
>> some WT cells survive through the drug selection. 20ug/ml of G418  
>> is used. Even for the real colonies growing up, sometimes only a  
>> few of them express target protein, others do not express but are  
>> G418 resistant. This will make me picking a stable overexpression  
>> cell line is as difficult as screening a knock-out strain!
>>
> again try longer selection (2 weeks) but there are other variables  
> too -    getting transformants also varies from gene to gene and  
> vector to vector, it may take longer too (like once it took almost a  
> month for me to get good % of overexpressor mutant). Also from my  
> experience i would say try to keep always in drug selection (until  
> you get good % of transformants and later you can keep them growing  
> at low concentration of drug) because when you take to KA plate  
> (where there is no drug selection) the cells tend to get rid of the  
> vector. So best is pick colonies directly from selection plate to 96  
> well plate.
>



If I may add to this: do not forget that if you do a very short  
selection (5 days is very short, especially for G418 selection), you  
select cells with a stable integration, but also MANY cells containing  
the non-integrated plasmid.  The latter are only lost upon prolonged  
selection as cells divide and the plasmid is diluted away.

Pierre Cosson
Centre Medical Universitaire
Dpt of Cell Physiology and Metabolism
1 rue Michel Servet
CH1211 Geneva 4, Switzerland
Email:[log in to unmask]
Tel. (41) 22 379 5293
Fax (41) 22 379 5338