3 replies to this topic

[andyg2886]

Hi all

I attempted to transfect a gene into human embryonic stem cells using the pCAG overexpression system. I had no luck overexpressing it, as mentioned in previous postings.

To see what was going on, I decided to do a DNA extraction from the cells. Cell lines include:

1. Wild type hES cell line
2. hES cell line transfected with pCAG-eGFP
3. hES cell line transfected with EWS gene (1)
4. hES cell line transfected with EWS gene (2)
5. hES cell line transfected with EWS gene (3)
6. hES cell line transfected with EWS gene (4)

I designed a huge range of primers to see whether it was endogenous EWS DNA being PCR'd out, or vector EWS. In my first PCR I designed a forward primer in the vector backbone, and the reverse primer in the EWS gene. Bands should only be present in the lines transfected with pCAG-EWS. This is exactly what I saw.

In a seperate PCR I designed forward primer spanning exon 6-7 of EWS and reverse spanning 7-8. Again, this should only bring bands out in lines transfected with EWS (as introns are present in endogenous DNA and so the PCR would not work for the endogenous gene).

This is not what I saw. I saw a band for this PCR in every one of my lines. Am I right in thinking this should only have brought out vector EWS DNA? Because that's not what seems to be happening.

Any help or thoughts on this would be greatly appreciated.

Cheers

Andy

[bob1]

Did you check that the primers are specific (primer blast)?

[andyg2886]

No, but the bands I'm getting in all lanes are exactly 200bp (what I designed the cDNA band to be). If there were other regions the primers were binding to, by chance these wouldn't be 200bp would they?

I have been thinking though... with a DNA extraction, I;ve been presuming that only genomic DNA (exons+introns) will be extracted. Am I wrong? If cDNA (exons only) is extracted too, then I would be expecting these bands in the wild type cell lines...

[bob1]

You are pretty unlikely to get any cDNA out of a DNA extraction - you might get some RNA though, but you would need to transcribe this to get cDNA.

If your primers aren't specific, you may amplify a 200 bp band, there's no reason why not.

It could also be that you have managed to contaminate on of your working solutions with PCR product (or plasmid with your gene) which would present as a contaminating band of 200 bp!