6 replies to this topic

[vgupta]

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?

[InvisibleSurfer]

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".

[tltan]

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

[mario2004]

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-...ingle_Cell_PCR/

Edited by mario2004, 16 August 2004 - 10:01 PM.

[amira]

tltan, on Aug 16 2004, 07:20 AM, said:

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

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

[marmal]

mario2004, on 16 August 2004 - 10:00 PM, said:

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-...ingle_Cell_PCR/

Hello Mario2004,

I am trying to do the same thing on HSC. I enclose a SNP in my studies to be sure that I get amplification of both alleles but often get amplification from one allele only. I digest my cell with 300ng/µl PK for 2h at 60°C in 0.5% NP40 and 0.5% Tween. Could you give me some advice how to improve my PCR? Which polymerase did you use?
Best regards
Marta

[Rsm]

I did a lot of cDNA library construction, multiplex qRT-PCR and mRNA subtraction from single primary cells, and I wouldn't do it again. It is not very reproducible... We followed a protocol described by Jensen and Watt (PNAS 2006). You essentially need lots of single cells (maybe 100) to get solid data, and if you need 3hrs to pick one, you'll think twice.

rsm