
single cell PCR
#1
Posted 11 August 2004 - 11:38 AM
Would anyone have a protocol for it and some trouble shooting tips?
#2
Posted 12 August 2004 - 02:01 AM
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".
#3
Posted 16 August 2004 - 06:20 AM
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
#4
Posted 16 August 2004 - 10:00 PM
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.
#5
Posted 18 August 2004 - 06:47 AM
You should just pick the colonie and put it in the PCR mix: it is very simple and works very wellHi,
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
#6
Posted 17 September 2010 - 07:07 AM
Hello 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/
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
#7
Posted 17 September 2010 - 08:11 AM
rsm