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?
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".
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
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/
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
You should just pick the colonie and put it in the PCR mix: it is very simple and works very well
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.
Agreed. My experience has been that its not necessary to do the extra sample prep steps.
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.
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/
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