Hi,

I have small frozen mammalian cell pellets (10-10000 cells) in a few uL of leftover media. I'm planning on doing some qPCR on the DNA using BioRad's iQ Sybr Green. I'm using 2 different sets of primers.

My plan is to resuspend the pellets in 100uL cold H2O and distribute 20uL x 4 wells into the plate wells with my master mixes. Then just hot start the reaction. My thought is that the starting 94C will break open the cells and inactivate DNAses & proteinases enough to start the reaction without first isolating the very tiny amounts of DNA.

Will this work?

Should I be adding something else to help it work?

Thanks for any advice.

Ed

Ed

You should look at this Kit

CellsDirect™ One-Step and Two-Step qRT-PCR Kits
Sensitive and specific, real-time qPCR results directly from cells

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-and-Expression-Profiling/qRT-PCR/Direct-from-Cells.html

Robust results - Real-time detection of targets from a single cell to 10,000 cells

Awesome,

Thanks! I will investigate this further and see if I can afford the kit!

Ed