Re: [DIYbio] Re: qPCR fluorescence detection dynamic range

I agree with Jeswin almost completely.

No, offense but I don't think anyone will use a DIY machine for diagnostic uses and telling people that it can be used that way is just looking for a disaster. (I tested X on the machine and it said I have X disease....)

One can detect all those things you mention with normal PCR.

You want to see if you PCRed something and it worked. People run agarose gels for that. Doing Real Time PCR to try and figure out if your PCR worked really seems like overkill. Doing it instead of sequencing,  probably costs 10x in reagents and 1000x in time.

Debugging gene expression. A much easier way is protein expression or Reverse Transcriptase PCR as Jeswin said.

The data Real Time PCR generates cannot usually be compared between experiments. It is kind of like a western blot, there is way too much variability. Real Time PCR really has not much place in biological engineering. What can one use it for? There are numerous papers that show that gene expression has no correlation with protein expression. People run SDS-PAGE gels or Western Blots. The variability is why most people tend to not quantify these things. Instead just say, X band is obviously more than Y which is greater than Z.

In qPCR you run a control of a known "housekeeping" gene such a GAPDH or Actin to have a relative comparison. The transcription of these mRNAs is dependent on minutiae like state in cell cycle or time "alive" or nutrients or temperature or microenvironment. Things that cannot be replicated easily not matter how hard you try. It is very bad form to compare one run to another run, same thing for stuff like Reverse Transcriptase gels or Western blots. The variability in mRNA expression makes it so only relative comparisons are possible. Error propagation in exponential processes is huge. 

Again we have not even gone back to the handling of RNA in a home lab.

I have worked with RNA plenty doing Ribosome profiling and the lab I worked in is on lockdown from touching anything without a gloved hand, ethanol wash everything before use. RNAse Away everything and still people have RNA degradation problems. I don't know how much faith I could put in data generated from a home lab with very few resources and many sources of contamination.

I don't mean to be so negative and I hate telling someone not to do something without giving them other options. So...

• I think your time would be better spent developing assays and protocols that are easy and cheap but can answer big questions. Why not setup a DIY bacterial two-hybrid system so in vitro evolution is easily available to people?
• Why not develop software for use in bioinformatics?
• A cool program would be to use machine learning to analyze conformational changes from molecular dynamics simulations using output from GROMACS programs.
• How ions actually find channels so fast i.e. how ions overcome the 3D diffusion random walk problem?
• Micromanipulation of DNA using AC and DC current ala http://www.sciencedirect.com/science/article/pii/S0006349502753988
• A non sequence based protein structure classifier using things like ramachandran angles, hydrogen bonds &c.

If you want to work on hardware

• An easy, cheap, DIY setup to make a light microscope a decent fluorescence microscope.
  
• A modern AFM(I know there is bunch of old stuff floating around on these things don't know about new stuff haven't looked it up in a while)
• A diode array or scanning spectrophotometer like what Nathan is trying to do. Maybe one that costs < $50 and has nice software and 2 nm or better resolution.

There are so many cool things and ideas to work on it seems like a waste of effort to put it into a Real Time PCR machine that wouldn't be extremely useful but in the end you should do what excites you the most even if other people think it is a bad idea.

On Thu, May 23, 2013 at 1:54 PM, Jonathan Cline <jcline@ieee.org> wrote:

Touch screen: how's that work with gloves on? Better make it resistive.  Except, no one likes those because they're really annoying to use (pressure, precision), especially now that we're all spoiled by capacitive displays.  Worse case you've got a membrane keypad (again, no one likes those) and a non-touch display.  Either way you'll have to compromise on display resolution and size, when you really want to graph something onscreen in a really large format.   Meanwhile one of the most successful equipment stories is still Nanodrop, which doesn't have a display at all, as I suggested: it sends all data to the nearby computer. 

Re: Wireless again.  Measuring very low voltages with sensitive electronics while beaming a bunch of RF energy all around right next to the amplifiers will cause trouble.  

Cheaper than competitive equipment?  Why not kill off the competing equipment with either a retail price so low that their margins are destroyed, or alternatively keep the "higher low" price as you suggest and keep more margin yourself?   Either way the cost of building the device should be lower, not higher.  

On Wed, May 22, 2013 at 10:41 PM, Josh Perfetto <josh@openpcr.org> wrote:

 I think that the touch screen is valuable for enabling proper use of the device/reaction setup in some use cases, and being a device which generates data, internet and web connectivity via ethernet/wifi are paramount to A) providing a great user experience for researchers to access and analyze their data, and B) enable others to build applications on top of the machine. By "low cost" hardware, I meant it would be cheaper than the cheapest generally available qPCR machine (which AFAIK is about $10k) by at least an order of magnitude, not that it would be the absolute cheapest machine possible.

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