What's wrong with open source PCRs

[Ujjwal Thaakar]

Hi,

I recently decided to design a PCR machine that's affordable since I can't either buy one or afford an OpenPCR. I wanted your views on OpenPCR, MiniPCR and the likes of others. Are these runaway successes? Did everyone really buy one for themselves? What were the issues you had with them? What does diybio need today the most and at what price point. 

--

Thanks

Ujjwal

[Bryan Jones]

Hi Ujjwal,

I bought an OpenPCR 3 or 4 years ago and have been very happy. It has generally worked great. I did have a temperature sensor go bad, but Josh Perfetto from Chai Biotechnology sent me a replacement for free (I think he is actually on this list). Myself and others in my academic research lab have used it regularly to do all kinds of experiments, including lots of site-directed mutagenesis, error-prone PCR, site-saturation mutagenesis, and to construct libraries using DNA-shuffling. I published some of my work last year citing OpenPCR (attached). I generally don't use our expensive mj research thermocycler at all anymore. 

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[John Griessen]

On 01/21/2016 04:25 PM, Bryan Jones wrote:
> Myself and others in my academic research lab have used it regularly to do all kinds of experiments, including lots of
> site-directed mutagenesis, error-prone PCR, site-saturation mutagenesis, and to construct libraries using DNA-shuffling. I
> published some of my work last year citing OpenPCR (attached). I generally don't use our expensive mj research thermocycler at all
> anymore.

How do the two compare?

[Bryan Jones]

How do the OpenPCR and the MJ Research machines compare?
The MJ machine cost us about 10 times more and has a bit more functionality. The advantages of the MJ machine has a higher capacity of 96 samples as opposed to 16 with the OpenPCR. The MJ can also do a temperature gradient so I can simultaneously test different annealing temperatures for different tubes. The MJ machine also did not require a computer. I think the MJ machine also has faster heat/cool speeds, so your run can be a little faster.

On the other hand, I like the OpenPCR interface a lot more. Since you use a PC, it's point and click, and type in the target temps and times. The stand-alone MJ machine uses an awkward interface with a hard to read display and unresponsive buttons.

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[Pieter]

We've compared openpcr and biorad mycycler years ago and there was no difference. OpenPcr only has difficulty with a low holding temp once the PCR is done

There's also NinjaPCR and GaudiLabs Wild OpenPCR, lightbulb pcr, and I believe the guys of syntechbio were also building their own design. So there is much to learn from and designs to try.

[Ujjwal Thaakar]

Yes. But another part of my question was how important is PCR to your daily usage? What improvements would you like to see. I'm planning to design something of my own and sell a few kits. Wanted feedback on what the community needs not just in PCR but other hardware and software.

[Ujjwal Thaakar]

Forget this thread. I just received a message from Josh Preffeto that I am trying to cheat him by subverting Indian customs and making a personal profit. That just makes me sad. I had asked for a rebate because customs is too high in India and I had little money then and I needed just 1 kit to understand how thermocyclers work because I have no experience in hardware whatsoever. I have lost all respect for him now. I had clearly said in the last message that I plan to design something of my own. It is just sad.

[Jonathan Cline]

Future lab devices will be USB 3.0 powered by 12V, no external power supply.  60W is enough for an efficient PCR machine aka thermocycler.  Build for the future, have it hit the streets in 2017 as fully powered by USB only. [But monitor market acceptance of the standard while developing the design.]  Check out this summary:

USB 3.1: Released in July 26, 2013, USB 3.1 doubles the speed of USB 3.0 to 10Gbps (now called SuperSpeed+ or SuperSpeed USB 10 Gbps), making it as fast as the original Thunderbolt standard. USB 3.1 is backward-compatible with USB 3.0 and USB 2.0. USB 3.1 has three power profiles (according to USB Power Delivery Specification), and allows larger devices to draw power from a host: up to 2A at 5V (for a power consumption of up to 10W), and optionally up to 5A at either 12V (60W) or 20V (100W). The first USB 3.1 products are expected to be available in 2016, and will mostly use USB Type-C design.

On Sunday, January 24, 2016 at 12:40:19 AM UTC-8, Ujjwal Thaakar wrote:

Forget this thread. I just received a message from Josh Preffeto that I am trying to cheat him by subverting Indian customs and making a personal profit. That just makes me sad.

Too frequently in open designs, when a branch occurs (that is what you are trying to do), the project originators get angry.  In some cases the branch far surpasses the original, and in some cases, the original subsumes innovations on the branch.  The originators should not get angry but that's beside the point.  Only by re-engineering human nature will that difficulty be eliminated.   OpenPCR was not cost optimized as concluded in previous discussions (even though it is marketed as a low cost PCR machine).  However consumable costs for experiments will far surpass the cost of the PCR machine so if you can't afford the current price, you're already in trouble aren't you?  This is not to say it's not a good project, this is to say, there may be better projects to focus effort on, especially, reducing the cost of the consumables [reagents] or optimizing a workflow which uses smaller quantities of consumables [such as sub-2 uL].


## Jonathan Cline
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[John Griessen]

On 01/25/2016 04:51 PM, Jonathan Cline wrote:
> /*optionally up to 5A at either 12V (60W) or 20V (100W)*/. The first USB 3.1 products are expected to be available in 2016, and

> will mostly use USB Type-C

100 Watts average is plenty for a small sample oven volume. If you wanted a super steep ramp up,
saving some energy in a super cap would let you. and it would let you operate when someone tries powering from
a backward merely 5V USB supply.

[Ujjwal Thaakar]

I think you're absolutely right about that. I don't see any reason to use a DC jack anymore. In fact I was thinking about USB-C for power. Haven't actually looked into USB 3.0 and 3.1. I'm not even sure exactly what they are. I am already looking to move beyond the 2012 technologies used in the open machines so far. I'm also thinking about IoT or control using a mobile app. Not sure if that makes much sense going forward. Will be figuring it out as I go.

[Ujjwal Thaakar]

Hi John. I didn't understand that.

[John Griessen]

On 01/26/2016 10:24 AM, Ujjwal Thaakar wrote:
> I'm also thinking about IoT or control using a mobile app. Not sure if that makes much sense going forward.

I like micropython -- it's ported to the ESP8266 low cost FCC certified wifi/microcontroller...and the new ESP32 will have
bluetooth also for phone app integration.

On 01/26/2016 10:25 AM, Ujjwal Thaakar wrote:
> Hi John. I didn't understand that.
>
> On Tuesday, January 26, 2016 at 6:49:27 AM UTC+5:30, John Griessen wrote:
> 100 Watts average is plenty for a small sample oven volume.

That means if the mass you are heating is small enough 100 Watts of power flow converted to heat by a resistive heater element is
enough to quickly cycle temperature -- enough to do PCR.

If you wanted a super steep ramp up, saving some energy in a super cap would let you.

That means using all of the average power flow available (using the 20 Volt 100Watt mode of USB 3.1 type C). Super caps charge up
quickly and hold energy densely, so they are a small practical component to store some energy between heater on times.
Then the stored energy can be released from the supercap to drive the heater harder when it is on.

and it would let you operate when someone tries powering from a backward merely 5V USB supply.

This means also using stored energy saved up between heater on cycles, but when the input is from a 5V USB source it could be 1.5A
or 3.0 Amps yielding 7.5 Watts or 15 Watts. That low power might limit thermocycling speed if cycling much mass in vials.
But, if you wait to charge the supercap for a bit, you could do a quick ramp up powered by the supercap.

[Ujjwal Thaakar]

That makes sense. Let me get back with some ideas.

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Thanks

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[Jonathan Cline]

The open lab devices don't use 2012 technologies.. they still use 1990's technologies, that's the thing that bugs many of us.  It's not only that they use an Arduino board and then have to employ an external Microchip ADC to do the actual analog work that bugs me.

Laws of thermodynamics means it is physically impossible to use normal 5V USB power for a typical thermocycler.  5W or 10W is not enough power.  1st law: "Can't get something for nothing."  Companies have produced "USB powered coffee mug warmers" before - these are scam products.  Regardless of ramp time, there must be enough energy (power) put into the system so the heat does not dissipate.  Buy one of these USB coffee warmer products and try it out for yourself if in doubt.  60W is possible for an efficient thermocycler but not sufficient for a large mass (heat block) like regardless of ramp time.  And you need more power to suck the heat back out in cooling.  Note: OpenPCR uses a 200W supply and the design uses a traditional large-mass block and still that block is smaller than most thermocyclers.  Two Hundred Watts, yes, capitalized.  First calculate out how to do proper heat transfer to/from PCR tubes within 60W of energy then design a device which satisfies that math (also accounting for losses).  Don't forget the anti-condensation lid.  Rather than reverse engineering the existing devices, start fresh with the hard math, and design something fresh which applies today's available technology.  

My bet is that USB 3.1 "20V 100W" won't happen mass market ever - there's no mass market need.  It might be used in a niche market for industrial control (products which will remain expensive because the industrial market is willing to pay).  Even if 100W is in the technical standards, people have to want/need a product for companies to have any drive or success building the product.  So that spec won't get built.  However "12V 60W" I bet could become mass market because of electric cars and smart/autonomous cars (automotive market is 12V).  But this will happen much later than the rest of USB-C rollout, maybe coinciding with broad adoption of smart cars -- which are still currently niche products.  I could be wrong on my bet which would mean it is a great bet to make against me.  You'd still need a 100W USB hub to power that single high-power 12V/60W USB-C port plus the two or three standard-power USB-C ports to round out a typical 4-port hub for a product, and this power has to come from somewhere - for example, automotive batteries if it's in a smart car (12V/15A accessory power = 180W).

The following has been repeated often enough in this group over the years that I call for a chorus  --  say it with me now:  5V/2A USB power is insufficient to power lab devices due to the laws of thermodynamics.  5V USB for a communications connection or small microcontroller power or a few LEDs, sure; heater power: no.

## Jonathan Cline
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[Jonathan Cline]

Does anyone else find it amusing that it's 2016 now and online forums
are still discussing how to build what basically amounts to a classic
internet-ready toaster?

Except that a PCR thermocycler doesn't even make good toast. Try it
with sourdough, it's a disappointment. Maybe that should be improved
too. Broaden the market appeal.

[Cathal Garvey]

I do feel that the problems we solve designing thermal cyclers are
equally applicable to, say, advanced kitchen appliances..so I reckon
it'd be cool to generalise the underlying control hardware and present a
common "core" for both lab and domestic equipment.

Aside from the fact that this would enable more rapid and horizontal
development, it would have the side effect that all those neat new
gadgets in a maker/hacker's kitchen would be readily converted to lab
equipment when we talk 'em around to DIYbio. ;)

[Simon Quellen Field]

Just so I understand the problem a little better (I have never used a thermal cycler before), let me ask some questions about the specs I have found for one.

The BIO-RAD C1000 Touch Thermal Cycler 96-Well Fast uses 850 watts of input power.

It has a maximum ramp rate of 5 degrees Celsius per second, and an average ramp rate of 3.3 degrees C per second. It holds 96 tubes, each with 0.2 milliliters of liquid in them.

Two other specs are a little confusing to me: the Gradient range is 30-100 degrees C,

and the temperature differential range is 1-24 degrees C.

So we are talking about heating and cooling 19.2 milliliters of water, and the mass of the plastic tube. From the graph on that page, it seems like a typical use case is cycling between 60 C and 90 C, at about 30 to 40 second intervals.

To be on the conservative side, I will assume that the plastic holder takes as much power to heat as the water, and I'll round up to heating and cooling 40 ml of material.

Raising 40 ml of water by 30 degrees C takes 5,020.8 joules.

Doing that at a rate of 5 degrees per second takes 6 seconds.

5,020.8 joules in 6 seconds is 836.8 watts.

Let's call that the brute force method.

Now suppose I have two water baths, one at 60 degrees, and another at 90 degrees.

I use a small motor to move the 96 tubes from one bath to the other.

I can have a small fan that blows over the tubes when they go from the hot bath to the cold bath to speed the cooling, so that when the tubes hit the 60 degree bath they are already at 60 degrees.

Now my heating problem becomes one of maintaining the 60 degree bath at 60 degrees (making up for the losses through the Styrofoam insulation, and the evaporative losses), and maintaining the 90 degrees bath at 90 degrees, making up for the losses in heating the tubes, insulation, and evaporation.

The 90 degree bath loses 5,020.8 joules every minute (cycling 30 seconds hot and 30 seconds cold). That's 83.68 watts. Plus a small amount needed to make up for evaporative and insulation losses. And a bit to run the fan. Call it 90 watts.

At 12 volts, that's 7.5 amperes. At 20 volts, it's 4.5 amperes.

The reason the USB3.1 specification calls for 20 volts at 5 amps is so it can be used to charge laptops and power monitors. I actually own a desktop computer with USB3.1 ports and Type-C connectors, so I may be able to give actual numbers, once I get a device that can charge through a Type-C port. But the manufacturer claims it can do 20 volts at 5 amps. You can get a PCIe card with a Type-C connector for less that $20 and try it out for yourself.

So, it should be just barely possible to build a thermocycler that runs off a Type-C connector. You don't even need to go the water bath route. You can put a laptop battery in it, and get the whole 850 watts while it is running, and charge it from the USB port when it is idle.

But realistically, how much more convenient is plugging it into a USB port compared to plugging it into the wall? Just use house wiring and throw power at the problem.

Having said all that, I think a cool project would be a single well cycler that heats and cools 0.2 ml of solution, powered by USB. That should be doable in 10 watts. :-)

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[Jonathan Cline]

Excellent point about a single-well thermocycler.  Rarely do you want to run 1 tube at a time however.  Need several tubes at a minimum, for experimental controls, redundancy, etc.  OpenPCR is considered personal-pizza-size with only 16.  Apple's USB-C laptop charger is rated at 29W ( 2A @ 14.5V ), for the Macbook which has no other power supply input, only USB-C -- so the supply isn't in either the 60W or 100W class.  Plastic tubes are unfortunately not very thermally conductive.  The liquid volumes are under 100 uL (typical synbio anyway).  I didn't check your math, I do agree that static temperature baths are the cheapest solution - either move the tubes to the bath or move the bath medium to the tubes.  Most of the energy goes into heating/cooling the metal mass holding the tubes, which oddly enough are machined out of large solid metal blocks -- there may be a benefit to this that I can't see, otherwise I consider it a baffling choice (no pun intended).  Some thermocyclers physically halve the heater block mass (I suppose you could call this, decoupling) when ramping temperature down.  Note the use cases may include incubation temperature for long periods of time (37 C typical, or up to 65C in some cases, for up to several hours total), this would result in steady state power draw, not peak power -- this is important regarding the assumption that a battery could be used instead (not sure I'd want to use a battery unless it was remote field use).  Another static temperature use case is 4C for many hours, for example.

The Lava-amp micro thermocycler was under 1W (I think).  Even had it worked out, I'm not sure it would have gained acceptance because it used a different form factor - didn't use "old fashioned" tubes.  Although there could be real experimental differences in results if changing equipment, obviously the ideal thermocycler would increase the surface area of the liquids for maximizing energy transfer -- i.e. not use plastic tubes.  That's one part of the approach the Lava-amp design took.

About this assumption:

>Now suppose I have two water baths, one at 60 degrees, and another at 90 degrees.
>I use a small motor to move the 96 tubes from one bath to the other.
>I can have a small fan that blows over the tubes when they go from the hot bath to the cold bath to speed the cooling, so that when the tubes hit the 60 degree bath they are already at 60 degrees.

Historically there are thermocyclers designs which use fans and electronically controlled vents to direct heated (or room temperature) air for assisted ramping.

Here's an example typical use case (synbio purification-ligation) to contrast to your example.

100 cycles	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
	12 °C	60 s
	22 °C	60 s
Hold	16 °C	infinite

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On 1/26/16 2:44 PM, Simon Quellen Field wrote:

[Bryan Jones]

My understanding is that PCR machines typically use solid metal blocks because they make it much easier to heat all the samples evenly and keep a steady temperature. The high heat capacity of the metal minimizes spacial and temporal fluctuations.

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[Sebastian S Cocioba]

Think something like this will work? Hand wrapped springs soldered to copper heatsink and thermal-paste-bound to two Peltier. Will at thermistors on sides. Pardon the messy job, cobbled it few nights ago. Will add a finned heat sinks to each and then a fan blowing from below that overlaps both heatsink like wind beneath gull wings. Will do some tests in the coming days. Two tubes, one control, one experiment. What y'all think?

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

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[John Griessen]

On 01/26/2016 09:09 PM, Jonathan Cline wrote:
> Historically there are thermocyclers designs which use fans and electronically controlled vents to direct heated (or room
> temperature) air for assisted ramping.

I think flowing air is the way to lower costs even though it has moving parts which will require engineering. The moving parts
could be organic piezo material for doors, teeny fan motors with long shafts, and be low cost possibly. Losing all metal blocks
will win out by air stirring.

>
> Here's an example typical use case (synbio purification-ligation) to contrast to your example.
>
>
> 100 cycles 12 °C 60 s
>
> 22 °C 60 s

> 12 °C 60 s
>
> 22 °C 60 s

> Hold 16 °C infinite


Is this below office/lab ambient, (requiring refrigeration), a frequent desirable use case?

On 01/27/2016 11:06 AM, Sebastian S Cocioba wrote:
> Think something like this will work? Hand wrapped springs soldered to copper heatsink and thermal-paste-bound to two Peltier.

Yes it will work, but not win the efficiency prize, or the speed of heat transfer prize either with the uneven coil surface.
Stirring the air between the peltiers would be better. The peltiers's masses will slow the ramp when you reverse and go from cool
to heat pumping.

If you could find a tiny motor/propeller from a toy battery powered boat to put in between there it would help. Trim the plastic
propeller to fit, and lose the coils of wire...

[Simon Quellen Field]

My favorite design (I think I saw it on this list) was the long plastic tube wrapped around a board, with one end of the board in hot water and the other in cold water. The fluid was pumped through the tube, and it alternated being in hot or cold water just by traveling around and around through the tube.

Hard to do with 0.2 ml samples, but it might make a fun science fair project.

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[Simon Quellen Field]

Looks like a fun project.

You can put the heat sinks in water baths for better efficiency.

Or put the hot one in an ice bath, and the cold one in hot water.

As we mentioned earlier, a thermocycler is an easy project if you throw power at the problem.

This thread was about getting the power requirements down to something a Type-C USB3.1 power supply could handle.

Not that I mind hijacking a thread that was close to played out. :-)

The cycles Jonathan mentions (100 cycles from 12 degrees to 22 degrees) is one of the easier ones to power.

12 degrees is a cold water bath, and raising the temp only 10 degrees every minute brings the power requirement down to under 28 watts.

This is one watt less than the Mac power supply provides through its USB3.1 Type-C connector.

How about this idea:

My washing machine is supplied with both a hot water tap and a cold water tap.

The cold water supply at my house is about 7 Celsius, and the hot water temperature is about 50 Celsius.

Hardware stores sell electronic sprinkler controllers that turn garden hoses on and off, using a microcontroller and a small DC motor to turn a plastic valve.

Now we just run hot or cold water over the 96 tubes and down the drain.

We could get fancy and adjust the temperatures the way the washer does -- by mixing hot and cold water in the right proportions.

The whole thing could run on a couple of AA batteries.

We aren't even talking about a lot of water. A trickle for 100 minutes. Less than a liter per minute.

With a pair of Y valves, you could even leave the washing machine connected, and just flip a couple valves to switch to the cycler.

Even the drain is right there. The cycler would just sit on the top of the washing machine when in use.

If you need more than 50 degrees, you could turn up the house hot water temperature, or add a small inline water heater.

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[John Griessen]

On 01/27/2016 07:45 PM, Simon Quellen Field wrote:
> The cycler would just sit on the top of the washing machine when in use.
> If you need more than 50 degrees, you could turn up the house hot water temperature, or add a small inline water heater.

It could work in a lab with sinks as well and it's a dirt simple concept. Might appeal to DIY as the most robust and maybe
lowest initial cost system if there are some generic and safe solenoid valves to use.

Is the "heated lid" feature needed to be "same as" the thermocycling temperature, or hotter to stop
condensation? "Same as" seems like it would be fine to me and as long as the water flow was surrounding
it would be "same as".

[Jonathan Cline]

Curiously, none of the supposedly open designers of previous PCR thermocyclers contribute their lessons learned from their previous projects.  Well it's not so curious perhaps, just greedy human nature, which even GPL attempts to exploit for it's own purposes.   Not so open, those self-proclaimed open people, are they?  At least not after even some meager dollar signs are seen.

The design you're thinking of was I believe the lowest tech copy of the Lava-amp or perhaps a hybrid.  It wasn't the first such design of it's kind, but a well publicized one. Lava-amp relied on thermodynamic mixing of liquids due to temperature gradients only, no pumping (thus much slower protocols too, I suppose), with three temperature zones (analogy: hot, medium, cold). 

One funny thing about mol bio experiments is that they are fragile in so many ways.  Yields are rarely (read: never) measured by biologists so it is difficult to characterize.  However some papers have noted experimental result differences depending on plastics used.  Surface area may play a role in that difference (chemistry in the liquid being biased by the plastics).  The last thing anyone wants during an expensive and time consuming protocol run is a drop in what I'm calling yield but could also be called quantity of viable biological product (protocols have too many already close-to-failure-mode variables, many of them unknown-unknown's).  Doing several protocols just to build something in order to build something later, which then later fails, is a massive head-scratcher and frustration builder.  This also in part leads towards the "let's stick with what humans have always done, to reduce changes, even though it's now an automated machine process, so let's use reaction tubes."

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On 1/27/16 5:21 PM, Simon Quellen Field wrote:

[Bryan Jones]

The plastic tube carrying the sample through different great zones is a similar concept to the "digital pcr" system from biorad. Just put the reaction in a bunch of micro droplets suspended in oil, in a really narrow tube, and added a florescence detector at the end and you have a digital qPCR system.

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[BraveScience]

Hi all,

@Ujjwal: that's a cool question. Personally, as it has pointed out above, connectivity is an issue. Even benchtop lab thermocyclers are kind of a pain to control smoothly. Actually they are horrible machines. Although I am part of the professional clients and my needs aren't always the DIYbioer's needs..

Price as well is challenging. There's a lot of technology to assemble and run properly and reliably. And price is very important for professional too.

All the suggestions so far pointed on making the system more efficient/cheaper and increased user experience (my point as well).

It's kind of remarkable, as well as happened for the fax, how thermocyclers didn't really change. They were horrible boxes and they still are. 

Like we weren't creative enough to come up with other systems that allow to heat/cool down some small amount of samples.

A good point was made by Jonathan: it's not too important to make a "cheaper" PCR machine by itself, but it's mandatory to reduce the amount of sample volume. What you can do in 50-100uL today you could easily scale down to 1-10uL. If you scale down to 1uL mix, well, it would be much much cheaper.

During colony PCR we usually use 10-20uL total volume. That means 5-10uL myTaq mix (all included). And it always works. I even used it for cloning, fuck those proofreading crappy polymerases. 

let's crunch some numbers: price tag is 104$ for 50uL reactions. That means 25uL master mix. Each reaction is 2$.

Going down by 10x means 5uL reactions would mean reaching 0.20$/reaction. Even in today's lab you can do quite a lot with 5 uL PCR volume if you reach a final yield of 100ng/uL. Enough to do plenty of cloning IF you avoid those horrible spin column/gel extraction kits... Gel extraction looses up to 90% of your stuff, especially if it >5kb.

Anyhow my point is size. Yet, and i know many will share this point, working with less than 10uL is a pain. 

If you work in a tube. 

Because pipetting isn't easy after the psychological barrier of <2uL and capillarity is a bitch.

Why don't we leave behind our backs those tubes? Come up with something new. 

Kind of muhammad story for molecular biology, if you cannot bring the heat to the sample, bring the sample to the heat. 

Samples could move across temperature gradients. You could get either low volume sizes and user friendly equipment.

Illumina and nanopore are already doing this. All sample preparations is ran on digital microfluidics biochips, although a patent fence as high as they sky is out there, and they perform an enriching amplification step previous to sequencing.

Best,

Fede

[Simon Quellen Field]

1. Patent fences should not bother DIY non-profit experimenters. Let them try to sue me for my $0 profit.
2. We have been discussing cheaper PCR machines. Perhaps we should be looking into cheaper consumables. At two million dollars a liter, there would seem to be some headroom on price. How about engineering an organism that produces the expensive part, and engineering a low-cost way of isolating it.
3. Is the expense of the consumables the main reason for going microfluidic? If larger amounts are easier to work with, and the consumables were cheap, would people bother with tiny volumes?

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[John Griessen]

On 01/28/2016 07:33 AM, BraveScience wrote:
> Why don't we leave behind our backs those tubes? Come up with something new.
> Kind of muhammad story for molecular biology, if you cannot bring the heat to the sample, bring the sample to the heat.
> Samples could move across temperature gradients. You could get either low volume sizes and user friendly equipment.
> Illumina and nanopore are already doing this. All sample preparations is ran on digital microfluidics biochips, although a patent
> fence as high as they sky is out there, and they perform an enriching amplification step previous to sequencing.
>

OK. Idea generating with sample not moving fluidly:
instead of vials,
use well plates with adhesive tape covers and flowing air for the heat flow medium

use pipette tips loaded, then dipped in oil to keep sample from evaporating and flowing air for the heat flow medium.
The thermocycler would look like something with wood pecker hole in it that you put the pipettor and tips into,
then press a button. (Would increase sales of pipettors that have a row of 6 or ten pipette tips -- do they work accurately?)



Idea generating with sample moving fluidly:

Hmm... that idea I just got was too good to blab about right now. This email list of thousands that don't speak at all is a
perfect way to just give away a nice IP kind of idea... Anyone wanting to go to Cork with me re: thermocycling droplet automation?

On 01/28/2016 10:13 AM, Simon Quellen Field wrote:
> Is the expense of the consumables the main reason for going microfluidic? If larger amounts are easier to work with, and the
> consumables were cheap, would people bother with tiny volumes?

I like your lateral thinking:-)

There IS always a reason to be able to process small amounts -- getting an inspiration from an accident where you only have a
little drop of it.

And ease of using hand held pipettors gets left behind when using automation -- then the repeatability and accuracy of machines
has no trouble with 2 uliters

[Jonathan Cline]

1. DIY for "$0" is fine but 98% of those even on this list just want to buy a ready-made product.  Myself included.  There are reliability and reproducibility and other -ity concerns if self-building custom equipment even if it is to a common spec.  It's mechanical engineering, not software.  The amount of time and effort to prep and run simple bio experiments can't be understated so having to build machines first is a no go.  Not even Woz sold many Apple I kits compared to how many ready-made computers he sold.   Can't standardize protocols and parts if everyone is using one-off custom builds.

Besides which, most DIY hardware designs are missing this critical aspect to them called:  *high priced engineering talent* which optimizes the design in part driven by cost constraints (capitalism works, ya know).

2. Cheaper reagents are being done and by some on this list but slow going.  And patent minefield is worse there.  DIY "grow your own" reagents are popular in research labs but again same concerns as #1, reliability and reproducibility and other -ity, when you buy a mix you are buying (supposedly!) the Q/A process not just the liquid itself.  Reliability in part is a weakness due to longevity (reagents expire and/or are  costly to maintain in a freezer), imagine if DIY computer hackers had to constantly re-generate their own silicon wafers to keep building new circuits..   And don't want people keeping reagents in their kitchen freezer next to their frozen vegetables and coconut ice cream (side note; sane biologists would not buy dairy ice cream).

3. Yes and Yes.  Sample density is still a reason to go microfluidic.  96 wells is okay for humans, 384 wells is hard to deal with by hand, 1536 wells is ridiculous.  The curve of available technology for data crunching means experiments should be run in "massively parallel numbers" yet one big limitation of this is simply the logistics of dealing with 1,536 separate experiments, whether handling them or tracking them.  As well as the desire to track the experimental effects on a single cell rather than a large culture of diverse and perhaps-behaving differently cells, that means, the environment and sensors have to be similar scale to the cell.

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[Jonathan Cline]

On 1/27/16 6:50 AM, Bryan Jones wrote:

My understanding is that PCR machines typically use solid metal blocks because they make it much easier to heat all the samples evenly and keep a steady temperature. The high heat capacity of the metal minimizes spacial and temporal fluctuations.

Yes, but wasn't that in the dark ages before today's machining could cheaply craft a perfectly shaped *hollow* form which could circulate a more thermally-conductive liquid into a much, much larger capacity mass i.e. external temperature bath?    For example:  newer very highly thermally conductive ceramic epoxies, cast as a PCR tube-shaped mold yet hollow to allow rapid fluid pumping to/from hot & cold baths would be very temp stable and physically light with faster ramp rates; note: temperature sensors embedded during the setting process to algorithmically control this.

Even temperatures across all samples is critically important.  Any thermocycler design must absolutely guarantee this within some very small % tolerance and the design must be verified to work within this tolerance.

On the rant about the use of outdated technology in designs, an analogy, tankless water heaters for home use (showers and kitchen faucets) have been available for some time, yet are still niche products despite their technology superiority, mainly because builders are either not aware of the benefits or are stick-in-the-muds.  So lab device technology is not so different from the same builders of a kitchen toaster in that way either.

[Nathan McCorkle]

On Wed, Jan 27, 2016 at 5:45 PM, Simon Quellen Field <sfi...@scitoys.com> wrote:

The cycler would just sit on the top of the washing machine when in use.

If you need more than 50 degrees, you could turn up the house hot water temperature, or add a small inline water heater.

What a great mental visual! Now purify those samples with the spin-cycle!!! Or alternatively, "the washer ate my homework!" (for those students out there). 

[Ujjwal Thaakar]

I really don't know what to make of all of this but I guess it's time to get down to some serious work and figure this out.

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[Simon Quellen Field]

I love the idea of a thin copper or aluminum plate stamped with tight-fitting dimples for the sample containers, and making a box with this as the lid, and running hot and cold water inside the box.

You get the temperature stability of a large water bath, and the rapid heat cycling without having to move the samples. Two large water baths kept at the two temperature extremes, and you just drain them through the box with the samples nestled in their dimples in the lid.

Spraying mineral oil or paraffin onto the sample containers before closing the lids also sounds like it would eliminate the need for heating the lids. So does the idea of simply filling the containers all the way to the top. No condensation if there is no vapor. The oil sounds a bit messy, though. At least paraffin is solid at room temperature. Why aren't the sample containers filled to the top? Even with distilled water?

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[Jonathan Cline]

You don't want to use mineral oil near the lids.  It is frequently noted as a band-aid to the problem that ends up being messy and possibly resulting in contamination.  It's also been mentioned previously in this group as well as a bad idea.

"Why aren't the sample containers filled to the top [such as with anything, ex. distilled water]"..  Umm.. because it's not beneficial from many angles - some Bio Lab 101 might be useful research here.  The purpose of PCR is commonly to amplify product as much as possible within a volume, so metaphorically watering it down is a bit opposite of the purpose.  Plus the experimental risk probabilities add up.  Pure water would add to reagent cost as well [not significantly if there's a steady source available, but still would add].   Ideally there would be a flux capacitor generating a neutron field which holds the target molecule in perfect chemically inert and isolated suspension while under operation by your chosen reagent, so the opposite of this ideal is an unknown chemical mix [liquid or solid] surrounding your target and interfering with it in random unknown ways and then unable to be removed later - you know what I mean?   The tiniest droplet which can later be removed 100% from the tube is best.

I believe John asked about the cooling use case (i.e. to 4C).  Yes it is a very necessary use case.

Where did the biologists go who want to explain these design criteria?  Hopefully they don't leave the fate of discovering the world's final new antibotic in the hands of an electrical engineer like myself, that only works in zombie movies.

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On 1/28/16 12:00 PM, Simon Quellen Field wrote:

I love the idea of a thin copper or aluminum plate stamped with tight-fitting dimples for the sample containers, and making a box with this as the lid, and running hot and cold water inside the box.

You get the temperature stability of a large water bath, and the rapid heat cycling without having to move the samples. Two large water baths kept at the two temperature extremes, and you just drain them through the box with the samples nestled in their dimples in the lid.

Spraying mineral oil or paraffin onto the sample containers before closing the lids also sounds like it would eliminate the need for heating the lids. So does the idea of simply filling the containers all the way to the top. No condensation if there is no vapor. The oil sounds a bit messy, though. At least paraffin is solid at room temperature. Why aren't the sample containers filled to the top? Even with distilled water?

 

[Mac Cowell]

That is how the first PCR machines worked, as I understand it Simon. Basically big hot tubs with the kind of baskets you see fries cooking in at a fast food joint robotically switching samples back and forth. Or, gasp, a poor grad student doing it manually.

I want to riff on your design, Simon.

Consider two water reservoirs: an insulated hot water carafe that keeps water near boiling (for tea etc) and a plastic cooler filled with ice water. 

The tanks are on a raised shelf. Each drains into an insulated silicone tube, say 1" inner diameter. The tubes connected with a Y joint. The base of the Y connects to a garden pump that pumps water back up to the hot water tank, which can overflow into the cold water tank.

Disconnect the tube from the base of the Y-joint and drop a ring of ~6 PCR tubes in, like shells into a revolver. Follow with thermocouple.

Fashion two servo-actuated hose clamps for the hot and cold tube. Differentially open/close clamps to set water temperature.

Beer-cave style PCR.

And a variant: if you're house's water heater is set to a dangerously high temperature, fashion a similar tube harness for a faucet and sink. Instead of hose clamps, use servos to control the faucet handles.

I wonder what the ramp rates would be.

Mac

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[Mac Cowell]

PersonalPCR arduino shield. low power requirements. Please hack.

https://github.com/100ideas/PersonalPCRv1

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[Mac Cowell]

Built worlds most dangerous hot air PCR thermocycler w/ Hackteria a while back. Arduino, triac, thermocouple, misc electronics, and 8-euro hot air hair dryer.

https://www.youtube.com/watch?v=XOjkyP8_Mm8

It was hard to determine the temperature of the mastermix in the tube. We tried embedding a thermistor in a similar mass at the bottom of a tube, but it was really fiddly. It seemed that the devil was in the details of knowing what particular temperature the tubes were at.

arduino code: https://gist.github.com/100ideas/1120142

It's interesting, I see maybe 5 different hair dryer pcr projects when searching google images for "hair dryer PCR"

One of them is from a 2015 iGEM team: http://2015.igem.org/Team:UMaryland/Hardware

Maybe we should have a contest: who can make the most unlikely / rube-goldbergy thermocycler work?

Keep on 'cyclin

Mac

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[Simon Quellen Field]

One problem with my idea is the cost of the water shutoff valves.

The battery powered ones are easiest to control with an Arduino or Raspberry Pi.

The cheapest I have found (with a cursory Google search) was $20 each ($40 total).

I'm sure that most of that cost is the computer and user interface (buttons and LCD), and that is the part we don't use.

If anyone sees a garden hose valve with electric shutoff, let us know.

The other option is to go with the $14 sprinkler valve, but it needs 24 volts, and some PVC conversion fittings to make it compatible with the garden hose / washing machine hose fittings. The 24 volt power supply adds to the cost, but can be switched with a cheap power transistor from the Arduino if we use DC (they normally use AC, but DC works fine).

Unfortunately, the cheap sprinkler valve is just on/off. The garden hose valve is run by a small DC motor and gears, and can be adjusted to any flow rate by timing how long the motor runs (stopping it before it is totally open or totally closed).

Washing machines have solenoid controlled water valves. I suspect they aren't cheap, or easy to remove from old washing machines.

eBay has some cheap solenoid valves, as does AliExpress.com.

I did find a motorized ball valve for $14 at AliExpress.com. But they want $8.35 for shipping.

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[Mac Cowell]

$20 is still probably cheaper than this 3D-printed servo-driven ball valve... But the servo could be handy. http://www.thingiverse.com/thing:1052421

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[John Griessen]

On 02/02/2016 11:41 AM, Mac Cowell wrote:
> It was hard to determine the temperature of the mastermix in the tube. We tried embedding a thermistor in a similar mass at the
> bottom of a tube, but it was really fiddly. It seemed that the devil was in the details of knowing what particular temperature the
> tubes were at.

that is a clue. Plastic is a heat lagger and also a heat sink with high heat capacity.
Changing to glass tubes would help. My kickstarter for an empty radio enclosure is not
inspiring maker hackers to instrument their back yards yet...

How many on this list would want a fast ramping air PCR thermocycler that needs no lid because it also
shakes the contents, (which also contributes to ramp rate performance)?

It could cost $190. On rev. 2 it might cost $120 and come with wifi webserving interface, python serial/USB port,
SD card for logging, 24VDC power port and wall wart power converter, and take up 10cm x 10cm bench space.

[Simon Quellen Field]

I'd support that.

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[Jonathan Cline]

The devil is not in the details. It's in the fact that you used a
thermistor.
Don't use a thermistor- that is basics.

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[Nathan McCorkle]

I've thought about this for a few years... it started while waiting in front of a drip coffee maker I think... I even bought some brass 12V valves from ebay, but I have never touched them (and there's not much biohacker scene here, I guess)

On Tue, Feb 2, 2016 at 10:36 AM, Mac Cowell <m...@diybio.org> wrote:
> $20 is still probably cheaper than this 3D-printed servo-driven ball
> valve... But the servo could be handy.
> http://www.thingiverse.com/thing:1052421

The $4 solenoid valves are $7 on AdaFruit, with the addition of a helpful voltage vs current table:

Voltage	Current
6V	160 mA
7V	190 mA
8V	220 mA
9V	240 mA
10V	270 mA
11V	300 mA
12V	320 mA

While they're recommending "a TIP120 or N-Channel power FET with a 1N4001 kickback diode to drive this from a microcontroller pin" I note that the voltage and current limit on a ULN2003 is 50V and 500mA... these are common driver chips that come with kits for LEDs and motors on ebay, etc... for driving such loads from a microcontroller/other sensitive source.

[Nathan McCorkle]

On Tue, Feb 2, 2016 at 6:05 PM, John Griessen <jo...@industromatic.com> wrote:

a fast ramping air PCR thermocycler that needs no lid because it also
shakes the contents, (which also contributes to ramp rate performance)?

Now you're encouraging/helping phase-change of liquid-to-gas... it would be worth checking for prior-art. Also shearing of the DNA, people might look at you cross-eyed if you so much as drop their sample tube, or mix it too fast with your pipette tip. You're going to have to confidently demonstrate you're not adding new variables to a protocol pipeline.

[Mac Cowell]

Anyone (simon hint hint) care to do a back-of-the-napkin estimate on the max ramp rate for tubes immersed in a flowing water thermocycler if the system can source 850 watts?

Simon, re: filling tubes completely with water - not what would happen; would the reaction just proceed more slowly in a more dilute system as long as the concentration ratios were maintained, or would it not work at all or be unreliable?

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[Jonathan Cline]

A microliter PCR thermocycler would be great at Chipotle restaurants.  They've lost millions in revenue due to their E. coli problem.  And supposedly they could not find the source of the bacteria.  (No, I don't eat there.)   If anyone here has theories on the topic or approaches to devices it would be neat to hear.


Quotes:

""In connection to the food scare, the company is now is facing an investor lawsuit in eight US states, lawsuits from individuals that contracted E.coli, and a federal criminal probe filed by the state of California.""
 http://www.bbc.com/news/business-35479980

" "Investigators were not able to identify the ingredient that was responsible for the contamination. Chipotle, which is based in Denver and has more than 1,900 locations, said it has already made many changes to tighten its food safety. The steps include moving the chopping of tomatoes and lettuce to a centralized location, and blanching onions to kill germs before they're chopped.""
 http://www.cnbc.com/2016/02/01/cdc-declares-chipotle-linked-e-coli-outbreak-over.html


I like that the article states that they're killing germs.  Valentine's Day is coming up, it'd be a bummer to get cooties. 

[Josh Perfetto]

Keep in mind that the primary purpose of a heat block is to promote thermal consistency between reactions. It may seem like a basic/old design, but it's a passive design that does an excellent job, like the wheel. The thermal mass of the heat block also plays a key role in controllability. Lack of these features was a detriment to several of the designs discussed previously in this thread.

As you've noted, increased heat block thermal mass does decrease ramp rates, and heat block design is paramount for both consistency and increasing ramp rates. Newer technology like additive manufacturing does enable new designs, but we haven't seen the costs become competitive with CNC fabrication yet, the costs of which have steadily declined in recent years.

People concerned about reducing PCR costs to the lowest possible levels should be taking a serious look at total costs including reagents. Thermodynamically the faster you want to go, the more enzyme and primer you need. I'm not saying we shouldn't work to do PCR faster, and one of my personal goals is to produce a device that can do 30-40 cycles in 1 minute. But if your goal is absolute cost minification, it's silly to be looking at ramp rates greater than 1-2 C/s.

-Josh

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[Sebastian S Cocioba]

https://www.streck.com/product.aspx?p=Philisa%20Thermal%20Cycler

Check it out. Uses what looks like squashed pipette tips as custom tubes. Peltier sand-which. Kinda like my wedge design. Apparently full 30 cycles in 11mins or so. Saw it in person. Crazy fast. My finger could barely keep up with the sensation of hot and cold when I kept it on the block! 

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CA%2BL%3DET2vkMpoeDun-aXf0S6HP78%3Dk%3DSi5%2B4Up0sQ%3D%2BT%2BrtLmUQ%40mail.gmail.com.

[Nathan McCorkle]

On Wed, Feb 3, 2016 at 11:52 PM, Sebastian S Cocioba <scoc...@gmail.com> wrote:

https://www.streck.com/product.aspx?p=Philisa%20Thermal%20Cycler

Check it out. Uses what looks like squashed pipette tips as custom tubes. Peltier sand-which. Kinda like my wedge design. Apparently full 30 cycles in 11mins or so. Saw it in person. Crazy fast. My finger could barely keep up with the sensation of hot and cold when I kept it on the block! 

Oh, neat! I've seen that one in a magazine, years ago, and even posted the image here on this mailing list (https://groups.google.com/forum/#!msg/diybio/yttYHgz0gFE/o4Icu_3--lwJ).

[Mac Cowell]

Just saw another open pcr project - "ninjapcr". OpenPCR fork. 

http://pcr.tori.st/index.php/Main_Page.  

https://github.com/hisashin/NinjaPCR

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[Cathal (Phone)]

I am very impressed with ninjapcr;

* Cheaper design
* Uses stock Arduino firmware!
* Has ninja in the name

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[Jonathan Cline]

"The thermal mass of the heat block also plays a key role in controllability."

This is because the old school control system is only second order.  When operating at faster speeds which seem unstable, the control system has to be upgraded as well.  Optimal filtering techniques in digital signal processing  (for example can be done on a $1.00 dsPIC chip) with 32 taps would take care of that- a real control system and with rapid response, and internally calibrated for unique manufacturing differences.  Much better than simple PID.  Temp sensors would have to be embedded to get accurate readings. 

Entire system cost to the end user has to be considered.  A lighter mass will be cheaper to ship for example.

## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
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On 2/3/16 11:11 PM, Josh Perfetto wrote:

[mobilebio]

Adding my $0.02 to this, as I've been away form the DIY scene for quite a while. A few years ago I attempted to construct the PersonalPCR machine using their open source documentation, none of which were functional at the time (note that it didn't say that in the literature...). No user support, and a lot of personal time came and went without a functional product at the end. I was upset for quite a while. The idea is great, and if someone were able to replicate it using the same concept, it's easily under $50 in parts. However, they now have a retail product that runs $650, and extensive software support for multiple platforms, including a phone. 

I just bought one, and am running it through its paces for the first time now. I won't probably get back here for a while, but it's an impressively simple piece of equipment. It's a thin film heating element attached to a low-mass aluminum block, which is cooled by ambient air. Heating and cooling times look to be in the 1.5*C/s range, which is fine for most things, and probably better than a lot of professional thermocyclers, honestly. 

So, basically, I'm over being upset about it not being open source. Unfortunate that they changed their model? Sure, but they have a good product, and because they're in the Boston biotech crowd, they'll have plenty of people to subsidize their development costs to 

It's a functional product that is a central part of DIY bio. Think of it as a startup cost, and then get to work on the fun stuff!

On Thursday, January 21, 2016 at 4:54:55 PM UTC-5, Ujjwal Thaakar wrote:

Hi,

I recently decided to design a PCR machine that's affordable since I can't either buy one or afford an OpenPCR. I wanted your views on OpenPCR, MiniPCR and the likes of others. Are these runaway successes? Did everyone really buy one for themselves? What were the issues you had with them? What does diybio need today the most and at what price point. 

--

Thanks

Ujjwal

[Dennis Oleksyuk]

What PCR did you buy?

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[mobilebio]

Ah, sorry -- the MiniPCR: http://www.minipcr.com/

It's the same as the personal PCR platform referred to in this thread, which no longer appears to have any documentation online. Shame.