PCR thermal cycler for DCG fixing

Dichromated Gelatin.
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Joined: Mon Mar 01, 2021 11:06 am

PCR thermal cycler for DCG fixing

Post by Erik » Mon Mar 01, 2021 1:32 pm

First, a big thanks to Jeff Blyth for the G307 thermal processing idea. I've found it to work great!

When processing a number of DCG exposures in a series, the manual steps of G307 processing can be tedious and interfere with throughput. Additionally, unless careful attention is paid, the manual timing and movement of plates through different temperatures is not as reproducible as desired.

A similar thermal process is used in biology for replication of DNA, the polymerase chain reaction (PCR). This involves heating a sample containing DNA to 95 C, holding it for a time interval, then rapid cooling to a lower temperature, holding it for a time interval, then repeating the cycle. Sounds a lot like G307! Due to the need for tight temperature control, devices exist to perform this processing rapidly and automatically, using peltier heating and cooling. These have been around for some time, and older models are ending up in surplus sales now.

Used thermal cyclers can be purchased online for a few hundred dollars.
I picked up a "for parts or not working" Techne Prime cycler on eBay, which turned out to have a minor electrical issue which was easily solved. Regardless, in terms of time saved in automatic processing, it is well worth it.

The thermal cycler is designed to accept a 96-well plate, which yields an effective heating area of about 80x120mm. My thermal cycler has a heated lid, which I set to 50 C. The heated lid and heated baseplate create a small oven, and are quite light-tight. I just place the plate directly on the grid meant to accept the 96-well plate, with the emulsion up.

The thermal profile I found to work best is very simple:
Heat to 100 C at the maximum rate, takes about 50 seconds to reach temperature from room temp.
Hold at 100 C for three minutes
Cool at maximum rate to 25 C, takes about 2 minutes to reach 25 C.
Hold at 25 C until the program is ended/restarted.

The fans of the thermal cycler are a little loud and would interfere with holography if running while an exposure is in progress in the same room. The fans turn off at the completion of the heat/cool cycle, however.

At some point, a further development would be to make a mold from a 96-well plate in sand or something, then cast the plate in metal to create a flat "tray" to put holo plates on, which would fit down inside the 96 wells and be in excellent thermal contact. Although, they say "if it ain't broke..."

Overall, I've found this to work quite well. I see posts from various users about how the manual steps of G307 can be a bit difficult to incorporate into a workflow, and thought sharing this automation may be of assistance to others.