ExtractionCellwallsGR

From 2010.igem.org

Extraction Cell Walls
During this process, keep all of your samples on ice before and after disruption (to prevent protease activity).

Wash all the parts of the '''french press style cell disrupter''' with demi water and 70% ethanol, 
put the collector and the pre-disruption chamber on ice for 10 minutes. 

Repeat this process in between disruption of different samples. (e.g. Bacillus and Streptomyces)

Dilute the harvested mycelium or collected pellet in demi-water 
about 30 plates/50 mL or 50/50 (volume) for bacillus pellets) and vortex for 1 minute.

Filter your samples using a 10 mL surringe and the filter mechanism provided (can be found at the french press).

A maximum of 15 mL diluted sample can be inserted into the pre-disruption chamber.

Disrupt your sample at 13 Kpsi.

Collect the disrupted cells using a clean surringe, then insert them into the chamber again.

Disrupt your sample at 13 Kpsi again, repeat this process 6x in total.

Collect you disrupted sample into sterile 50 mL Greiner tubes, make sure you collect as much as you can, 
from both the collection chamber as well as reminants from the pre-disruption chamber.

Put your collected samples on Ice!

Spin down your samples for 5 minutes at 5000rcf.

Cook your samples in 2% SDS

Spin down your samples for 5 minutes at 5000rcf.

Cook your samples in 2% SDS

Wash your samples 10x with demi-water.

Freeze your samples (-80, 20 minutes minimum) 

Dry-freeze your samples over night.