4 replies to this topic

[paramyosin]

Hello all,
I am a beginer with mammalian cells transfection and i have tried with the expression vector pIRESneo (although i am not managed to obtain big expression levels). When i obtain surviving cells in presence of the antibiotic G418, i try to isolate the plasmid in order to perform a PCR to amplify mi heterologous gene. The fact is that i did not obtain anything at all in several times i have performed a traditional isolation of plasmid DNA. I have several questions: do anyone know the way of working of pIRESneo?, it maintains itself extrachromosomaly or it is included in the genome? (i wasn´t able to find any information about this issue), and a traditional plasmid DNA isolation can be performed succesfully in mammalian cells to recover plasmids?, if not, any of you could send my a web page or a protocol to obtain plasmid DNA from this cells?, the protocol that i am using is the E. coli traditional one with Glc buffer, acetate, phenol extraction and ethanol precipitation.


Thank you in advance!

[fred_27]

Hi paramyosin,
when you stably transfect cells (http://en.wikipedia....nt_transfection), the gene integrates into the cells genome. Due to selection pressure (iresNEO) it's not lost during cell divisions.
Therefore it is in this case not possible to isolate extrachromosomal DNA (for isolating stable extrachromosomal plasmids use the method established by HIRT / if you need a protocol I may look, wether I find my old one) but you have to isolate genomic DNA and to PCR.

Fred

[paramyosin]

Thank you Fred, yes, it would be very useful for me this protocol if you find it, just in case i woul need to isolate extrachromosomal DNA from mammalian cells.

Thank you very much for your pront answer

[fred_27]

Hi paramyosin,
this protocol worked for me (I found it somewhere in the internet). I qantified the DNA with qPCR. Unfortunately I didn't found the original paper. But I think it was:
Selective extraction of polyoma DNA from infected mouse cell cultures.
Hirt B., PMID: 4291934.

Fred

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[Dr. Desai]

paramyosin, on 07 May 2009 - 01:28 AM, said:

Hello all,
I am a beginer with mammalian cells transfection and i have tried with the expression vector pIRESneo (although i am not managed to obtain big expression levels). When i obtain surviving cells in presence of the antibiotic G418, i try to isolate the plasmid in order to perform a PCR to amplify mi heterologous gene. The fact is that i did not obtain anything at all in several times i have performed a traditional isolation of plasmid DNA. I have several questions: do anyone know the way of working of pIRESneo?, it maintains itself extrachromosomaly or it is included in the genome? (i wasn´t able to find any information about this issue), and a traditional plasmid DNA isolation can be performed succesfully in mammalian cells to recover plasmids?, if not, any of you could send my a web page or a protocol to obtain plasmid DNA from this cells?, the protocol that i am using is the E. coli traditional one with Glc buffer, acetate, phenol extraction and ethanol precipitation.


Thank you in advance!

I think you can also do the PCR directly using the cells, as we do in colony PCR. You just need to take the no. of cells,and add the gene specific primers and do the PCR which tell you whether your gene of interest is integrated in your cells or not.

Preeti