single cell PCR - (Aug/11/2004 )

Has anyone tried single cell PCR? Have you faced any problems with it?
Would anyone have a protocol for it and some trouble shooting tips?

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you mean pcr-ing from a single colony?

If so, I've done it several times and it works pretty good.

Simply pick a colony, resuspend it in say 20-50ul of Tris pH8, boil 100'C for 3-5min and use that as the target DNA in your PCR. I usually use something like 5-10ul of that "boiled colony".

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Hi,

I always work on the concept of 1 colony per 20ul of sterile H2O.
resuspend the colony (if thick) by vortexing or pipetting. Then heat as per mentioned and cool it down immediately. To avoid contamination, do a spin to pull down the cell debris

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I think vgupta really meant single cell PCR, that is to use a single cell as the template for PCR amplification of whatever DNA, RNA.

I have never done single cell RT-PCR, but I have tried to isolate single cell from culture using a glass capillary, digest the genome DNA using methylation sensitive enzyme and do a PCR to detect DNA methylation. It is not a big deal.

There is a protocol here: http://www.protocol-online.org/prot/Molecu...ingle_Cell_PCR/

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You should just pick the colonie and put it in the PCR mix: it is very simple and works very well

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could you tell me the exact nember of the cell used to PCR. Is there any differences in enzymes between the common PCR and the cell PCR .thanks lot.

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Agreed. My experience has been that its not necessary to do the extra sample prep steps.

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HI there,
if you're really looking into single cell PCR, there is a specialized company out there called Arcturus. They're specialists with the laser capture technique in both equipment as kits. No problem with single cell rna isolation and pcr-ing for me. Good Luck.

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hi mario,

I couldnot open the site you ve mentioned. I am trying to do single cell PCR of pulmonary artery smooth muscle cell. I am facing lots of problem. has anyone tried to pick up a single cell and perform Rt-PCR. if so please suggest.
liza

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