I need to turn solid insect tissue (thorax muscle) into a cell suspension. This is required to then embed the individual whole cells in a gel for a "gel plug" extraction of long DNA molecules (hundreds kbp) for optical mapping. The insect has no liquid inside to speak of, so I need to turn thorax or abdomen tissue into a suspension of individual cells, without breaking up the cells and shearing DNA. Is there a modern protocol for this?
A simple approach could be to grind the solid insect tissue by freezing it in liquid nitrogen and then crush the tissue by grinding it. This will yield a mixture of broken tissue and cells, but many cells will remain intact including the DNA. This will also require optimization steps for how much grinding is good, and if other grinding enhancers such as glass beads may help. This is not a modern protocol but could become one.
Dear Shahid,
Thank you, but this seems like a bad idea. Grinding would result in breakdown of some cells and some DNA molecules. When we transfer the cells to the gel plug for HMW gel extraction we will have a mix of sheared and intact DNA molecules, but the goal is to have longest DNA possible and not shear. I need a gentle method of dissociating cells without breaking them.
You need to separate the intact cells from the ones that have broken DNA.
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