Filter and Circulator settings

Andy_Donaldson

Hello, I have just a few quick questions regarding the buffer filter and the settings for the water circulator during the ETC step. First of all, how often do you replace the filter cartridges? I am in the process of assembling my apparatus and I'm not sure how many replacement cartridges to order. Secondly, what level do you have your pump flow/pressure at during ETC? Do you change out your clearing solution at any point during ETC? Finally, did/does your circulator have any problems with circulating the clearing solution? Thanks so much for your help.

joelrosiene

CLARITY FAQ released a few days ago says after every 2 whole brains - nothing beyond that. In my rig, I have been running the same filter for perhaps 3 brains now - but it probably depends on the rates of clearing, and of the 3 brains I have attempted, only one has been a partial success.
http://clarityresourcecenter.org/docs/CLARITY_faq_6-16-13.doc

In terms of replacement cartridges - I have 5 in a drawer here. They aren't terribly expensive.

Flow rate - I am using a cheap fish tank water circulator in place of the fancy standup equipment. I have noted that any flow through the chamber should be kept in the range of 3ml/second or less, and agitation of the sample should be minimized, as after a few days pieces of gel begin to break off.

My circulator, cheap as it is, has no problem at all pushing the solution through the filter and the chamber circuit.

MNG

I am doing the same thing with the fish pump, but got a black precipitate on the cathode and tissue. All the hosing is clear though, making me think it came from the pump. Which brand of pump do you use and have you had a problem with crud collecting anywhere?

joelrosiene

MNG I have had the exact same issue. The precipitate does not seem to interfere with the electrical properties of the cathode, however, as my amperage remains within expected ranges. After examining my particles under the scope, I do not think they are plastic pieces. My best bet is pieces of gel or other contaminant that have been oxidized by the current. These pieces tend to build up on the sample.

I am running a lower voltage at this time (20V), and at 37C in an attempt to prevent this from happening again.

I have also noted that if metals other than the platinum cathode are exposed to the current, you end up with significant corrosion and oxidation after just a day or two. To prevent this, I have coated all non-platinum metal components of my setup with Lexel, which is a natural rubber silicone alternative. This seems to be helping.

At one point I had significant buildup of a blue-green copper residue that also seemed to love the gel. Made for a cool picture, but obviously very bad imaging Again, the Lexel took care of it.

The pump is a ZooMed TCN-30, if that helps.

(Edit):
Don't use teflon tape to waterproof. The Teflon is nonreactive, but plumbers teflon tape is coated with wax - which gets into solution, and cakes up on your sample just like the black crud.

Andy_Donaldson

Question about the fish tank water circulator: how much clearing solution does it hold? Do you change out the clearing solution at any point in time during the ETC step? Thanks!

joelrosiene

The tank itself hold about 500ml, the entire setup? perhaps a liter and a half. I don't change solution during the ETC step. Maybe after you do a few brains. The FAQ suggests that the main reason to change solution is when the pH drops as a result of the electrophoresis. They suggest changing solution when the pH falls from 8.5 to below 7.

Andy_Donaldson

Thank you. One more question: when using your fish tank pump, how do you heat your solution to 37C? A water bath maybe?

joelrosiene

Here are a few pictures of my setup:

https://docs.google.com/file/d/0B6RE82086JGFT1gxYlZldnE4Vlk/edit?usp=sharing

https://docs.google.com/file/d/0B6RE82086JGFRzBDa1U2SFQ4Y3M/edit?usp=sharing

https://docs.google.com/file/d/0B6RE82086JGFNWE5c0k2NW5mVTA/edit?usp=sharing

Pardon the clouding of the clear PVC - looks like the schedule 80 isn't up to handle the heat coming off a 94V run :-)

There you can see filter, with circulator behind it, and the water bath they are in.

Sorry for the mess.

liz_kritzer

To chime in, I'm also using a cheap setup consisting of old equipment we had hanging around the lab. I use a hybridization bath to heat my solution (4L total volume) to 37C. According to the temperature sensor in the bath, it's pretty stable. I imagine you could use any water bath.

Andy_Donaldson

Thank you so much for the pictures and your help!

gjones

We are currently just using an old (ancient) electrophysiology perfusion pump that really seems to do the job. We also had the issue of deposits on the cathode at the upper limits of the voltage recommendation, but just switched the cables to clear it off and then run all of our clearing at around 30V. There's never been any precipitate since.

MNG

We've been taking a page from the DIY sous vide stuff (coffee heater, PID and SSR) as well as finding cheap power supplies from China. We've also used .2mm wire on a frame for the electrodes. The lab across the hall bought the list out right and I was told it was in the 12 -15 K range. We would like to get this down to less than 1K for a fully functioning single unit, allowing for increased parallel processing (10 units, perhaps more if we put multiple chambers in parallel for a given pump/power supply). We're also working on an open "waterfall" type chamber that will be open topped and pour back into the bath.

After our next run, we'll post additional details as to what worked and didn't.

Just to confirm others' experience, our first run was at 50C and 60V (highest part of the range mentioned in the paper). Totally melted the brain and even caused some heated the wire to melt into the plastic on the chamber and break a solder joint. -- lesson learned.

Good to know about the teflon tape

Vortexam

@ joelrosiene what is material of your chamber made of?PVC or Polypropyelene?