Re: [DIYbio] Re: Low cost PCR(under development)

We have a prototype Open qPCR machine at the moment which is capable of
ramping ramping 5-10 C/s at various parts of the temperature range. Final
controlled ramps in the PCR region are likely to be more like 5-7 C/s. The
main difference over the OpenPCR was much higher power density in the
thermoelectric modules, which necessitates larger power supplies and higher
current power switching components. Another difference was reduction in
thermal mass of the heatblock by removing more excess material. A third
change was a faster heat exchange rate with the air when cooling (i.e.
larger heatsink and faster airflow). As Cathal pointed out this requires
more engineering in heatblock consistency and thermal control.

All of this increases cost relative to the conservatively engineered
OpenPCR. The increased cost may not be worth it for everyone, but I think
there is certainly benefit to PCR cycles that take 30 minutes instead of 2
hours. Anyways hope this answers your question about how faster ramp rates
are possible in heat block based thermocyclers. There are some more exotic
designs that achieve even faster ramp rates with high speed airjets.

-Josh


On Mon, Jun 30, 2014 at 7:30 PM, Josh Perfetto <josh@chaibio.com> wrote:
We have a prototype Open qPCR machine at the moment which is capable of ramping ramping 5-10 C/s at various parts of the temperature range. Final controlled ramps in the PCR region are likely to be more like 5-7 C/s. The main difference over the OpenPCR was much higher power density in the thermoelectric modules, which necessitates larger power supplies and higher current power switching components. Another difference was reduction in thermal mass of the heatblock by removing more excess material. A third change was a faster heat exchange rate with the air when cooling (i.e. larger heatsink and faster airflow). As Cathal pointed out this requires more engineering in heatblock consistency and thermal control.

All of this increases cost relative to the conservatively engineered OpenPCR. The increased cost may not be worth it for everyone, but I think there is certainly benefit to PCR cycles that take 30 minutes instead of 2 hours. Anyways hope this answers your question about how faster ramp rates are possible in heat block based thermocyclers. There are some more exotic designs that achieve even faster ramp rates with high speed airjets.

-Josh


On Mon, Jun 30, 2014 at 4:05 AM, Cathal Garvey <cathalgarvey@cathalgarvey.me> wrote:
There are two ramp rates; ramp while increasing, and ramp while
decreasing. Most machines can achieve 3-4C increasing because they just
use heating coils, but their decreasing temperature (accomplished with a
peltier, cooling fluid, etc.) may be lower.

IIRC, OpenPCR has a climbing ramp rate of 2-3C, and a reducing ramp rate
of 1C. Larger machines with fundamentally the same architecture (coil +
peltier) achieve higher rates merely by adding extra wattage.

Remember though, an additional consideration is how homogenous the
temperature change is through the block, and at what rate the
temperature change in the block is reflected in the tubes. There would
be little point doubling the ramp rate of the block if the tubes cooled
at an only marginally faster rate. Worse, doubling the ramp rate as
measured by the thermometer may look great on the charts and benchmarks,
but the block may be unevenly cooling and this can have real
consequences when you're expecting your samples to experience identical
conditions.

It's rare that you'll need decreasing ramp rates faster than 1C, in any
case. Have you a special experiment in mind?

On 30/06/14 10:59, Michael Shamberger wrote:
> OpenPcr has a ramp rate of 1 degree C/s.  I checked specs for some
> commercial versions and they have ramp rates of 3 or 4 degrees C/s.   What
> are they doing differently?
>

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Re: [DIYbio] Re: Low cost PCR(under development)

Yep, think about a hair drier. I also have a hot air PCR
thermocycler... it uses a light bulb for a heater... but didn't seem
to have a great ramp rate.

For modifying a hair drier heater coil to be arduino-controllabe,
these two instructables should be useful:
http://www.instructables.com/id/Extreme-Surface-Mount-Soldering/step1/Order-Parts/
http://www.instructables.com/id/Closing-the-Loop-on-Surface-Mount-Soldering/

they don't include snubbers, so couldn't be used to control the fan
motors, but it will work for the AC heater component which is going to
be higher amperage than the fan(s) anyway.

On Mon, Jun 30, 2014 at 6:48 PM, Cory Tobin <cory.tobin@gmail.com> wrote:
> Actually, I take back what I said about air not transferring heat to
> the sample very quickly. I remember a device a long time ago, I think
> it was called the "Indee" or something like that, that used really hot
> air and circulated it around the tubes really really fast. I think
> the ramp rate was around 8C/s. They basically made up for the poor
> thermal conductivity by heating the air up to like 200C and then
> bringing it back down as the calculated sample temperature reached the
> target temperature. Which made it faster than the peltier devices.
> So maybe air wins out in both cost and speed if designed right.
>
> -cory
>
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Re: [DIYbio] Re: Low cost PCR(under development)

Actually, I take back what I said about air not transferring heat to
the sample very quickly. I remember a device a long time ago, I think
it was called the "Indee" or something like that, that used really hot
air and circulated it around the tubes really really fast. I think
the ramp rate was around 8C/s. They basically made up for the poor
thermal conductivity by heating the air up to like 200C and then
bringing it back down as the calculated sample temperature reached the
target temperature. Which made it faster than the peltier devices.
So maybe air wins out in both cost and speed if designed right.

-cory

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Re: [DIYbio] Re: Low cost PCR(under development)

Maybe I'm missing something, as I haven't been following this thread from the beginning, but if your heater has to heat and cool the aluminum as well as the sample, the problem you are trying to solve is exacerbated, not alleviated. If you are moving the sample from a hot aluminum block to a cold one, then you are fine. Otherwise, you'd want low thermal mass, and air is pretty good. So would be a design that moved hot and cold water into the space around the sample. The water is a good heat conductor, and it is easy to keep large reservoirs of water at a constant temperature, and then move small amounts of water at the proper temperature into a chamber full of samples. Two valves, and gravity can move the water from the reservoirs through the device and into the sink.
Pretty cheap.



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On Mon, Jun 30, 2014 at 5:45 PM, Cory Tobin <cory.tobin@gmail.com> wrote:
> Having a non-block with zero mass is even better.

Sure, except that air doesn't transfer heat to your sample nearly as
fast as aluminum.  Imagine putting your hand in a 100C oven vs putting
your hand on a 100C frying pan.  That's why most thermalcyclers take
into account the volume of the sample and calculate the temperature
instead of just using the temperature of the block.

I'm not saying air won't heat the tubes sufficiently fast.  If you are
optimizing for cost then air is the way to go.  But heating and
cooling the air rapidly won't necessarily lead to the temperature of
the sample being heated/cooled faster compared to aluminum.

-cory

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Re: [DIYbio] Re: Low cost PCR(under development)

> Having a non-block with zero mass is even better.

Sure, except that air doesn't transfer heat to your sample nearly as
fast as aluminum. Imagine putting your hand in a 100C oven vs putting
your hand on a 100C frying pan. That's why most thermalcyclers take
into account the volume of the sample and calculate the temperature
instead of just using the temperature of the block.

I'm not saying air won't heat the tubes sufficiently fast. If you are
optimizing for cost then air is the way to go. But heating and
cooling the air rapidly won't necessarily lead to the temperature of
the sample being heated/cooled faster compared to aluminum.

-cory

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Re: [DIYbio] Re: Low cost PCR(under development)

On Mon, Jun 30, 2014 at 5:24 PM, John Griessen <john@industromatic.com> wrote:
> Are you meaning measuring temperatures with fine precision inside or next to
> the
> sample vial and comparing heat flow from the heater source by Watts
> delivered?

Not too sure... I feel like the sensor would either be inside the
liquid or maybe non-contact IR? I believe the idea is you command
constant power to the heater (or maybe a constant increase in power),
then watch the temperature, and look for when the temperature curve
isn't smooth. Then do the same thing in reverse cooling the sample.

>
> The usual plastic vial might get in the way -- maybe it should be silicon
> glass,
> and the sample size would have to have more mass than usual, or the machine
> would have to have
> super insulation. Sounds non-low-cost at first glance.

Not too sure.

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Re: [DIYbio] Re: Low cost PCR(under development)

On 06/30/2014 11:04 AM, Dakota Hamill wrote:
> It can't hold 96 samples, and requires oil due to no heated lid,

Heated lids are not essential. Absence of unwanted heat flow is.
My concept will do fast heat flow and even heat distribution without
a separate hater in the lid.

The "lid" won't look like a lid, and will not be near the sample vials.
The swirling air will be near the sample vials.

On 06/30/2014 01:26 PM, Cory Tobin wrote:> the tube block has a lower mass
> than the OpenPCR block.

Having a non-block with zero mass is even better.

On 06/30/2014 06:35 PM, Nathan McCorkle wrote:> do some neat target detection stuff with something like
> differential scanning calorimetry

OK... melting and glass transition phase changes...

Are you meaning measuring temperatures with fine precision inside or next to the
sample vial and comparing heat flow from the heater source by Watts delivered?

The usual plastic vial might get in the way -- maybe it should be silicon glass,
and the sample size would have to have more mass than usual, or the machine would have to have
super insulation. Sounds non-low-cost at first glance.

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Re: [DIYbio] Re: Low cost PCR(under development)

On Mon, Jun 30, 2014 at 4:35 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
> You transition through all the other possible molecular states faster,
> all the possible base-pairing of a primer to a target sequence
> (avoiding sequences that don't match the best)...

(for the specified temperature end-point)

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Re: [DIYbio] Re: Low cost PCR(under development)

On Mon, Jun 30, 2014 at 8:48 AM, John Griessen <john@industromatic.com> wrote:
> On 06/30/2014 06:05 AM, Cathal Garvey wrote:
>>
>> t's rare that you'll need decreasing ramp rates faster than 1C, in any
>> case. Have you a special experiment in mind?

I'd say that you always want faster, unless you want your PCR to be
sloppier. Sloppiness depends on a lot of factors, ramp rate is one,
but a properly designed primer is another.

>
> Are there some good research reasons to go for more delta T rate?

Yes, it gives the molecules less time to be random and jiggle around.
You transition through all the other possible molecular states faster,
all the possible base-pairing of a primer to a target sequence
(avoiding sequences that don't match the best)... the math is probably
something about the speed of the temperature delta being closer/faster
than than the speed of diffusion... thought there are the electronics
of basepairing too, but that'd be where I'd generally start to think
about a model of this.

> My stirred air cooled PCR cycler concept is likely to do more
> when I get around to doing it and testing it. And for evenness,
> it will be top performing inherently.

Yep, instantaneous with nice power metering, and you can probably
start to do some neat target detection stuff with something like
differential scanning calorimetry (maybe pair this with qPCR?)
http://en.wikipedia.org/wiki/Differential_scanning_calorimetry

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Re: [DIYbio] Re: Low cost PCR(under development)

On Mon, Jun 30, 2014 at 4:05 AM, Cathal Garvey
<cathalgarvey@cathalgarvey.me> wrote:
> Remember though, an additional consideration is how homogenous the
> temperature change is through the block, and at what rate the
> temperature change in the block is reflected in the tubes. There would
> be little point doubling the ramp rate of the block if the tubes cooled
> at an only marginally faster rate. Worse, doubling the ramp rate as
> measured by the thermometer may look great on the charts and benchmarks,
> but the block may be unevenly cooling and this can have real
> consequences when you're expecting your samples to experience identical
> conditions.

I'd say that uneven heating or cooling will produce a thermocycler
that will be quite hard to use for normalizing experiments... you'd
never be able to run things in parallel.

The fastest ramp rates I've seen is 15C/sec heating, 12C/sec
cooling... see the pic of an advertisement here:
https://groups.google.com/d/msg/diybio/yttYHgz0gFE/o4Icu_3--lwJ

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Re: [DIYbio] Re: Low cost PCR(under development)

> What are they doing differently?

In addition to what others have said, the tube block has a lower mass
than the OpenPCR block. The OpenPCR block is a solid block with holes
drilled in it. If you look at newer machines from BioRad, Thermo,
etc, they have designed the block to have a much smaller mass while
having the same surface area in contact with the tubes. See this
picture: http://www.bio-rad.com/webroot/web/images/lsr/products/amplification_pcr/product_overlay_content/global/lsr_cheese_block_expand.jpg
But as far as I know, shorter ramp rates just means shorter run time.

-cory

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PRIORITY : Seeking a SQL Server DBA who has a GC or US Citizen - skype hire.

Hello,           

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Re: [DIYbio] Re: Low cost PCR(under development)

Well the Idaho Tech halogen light / vortex air thermal cycler which was the first one i got off ebay has one major advantage over any peltier device I've used - it saves a boatload of time.  The ramp rates are much faster, especially the decreasing, as a vent is just flips open and hot air pours out.   It really cuts the PCR program almost in half on time, though I should actually measure it, but it generally finishes a good 30-45 minutes before a peltier device doing a 30x program, which normally takes ~1.5 hours.

It can't hold 96 samples, and requires oil due to no heated lid, but it's worked before and the time it saves more than makes up for those other disadvantages for the uses at the moment.  Obviously being able to run a lot of samples in parallel with a temperature gradient for annealing temp without oil is where the peltier machine is better but, it's also a new machine.  There are plenty of old peltier based devices without heated lids with crappy ramp rates, and for that I prefer the lightbulb machine.


On Mon, Jun 30, 2014 at 11:48 AM, John Griessen <john@industromatic.com> wrote:
On 06/30/2014 06:05 AM, Cathal Garvey wrote:
t's rare that you'll need decreasing ramp rates faster than 1C, in any
case. Have you a special experiment in mind?

Are there some good research reasons to go for more delta T rate?
My stirred air cooled PCR cycler concept is likely to do more
when I get around to doing it and testing it.  And for evenness,
it will be top performing inherently.


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Re: [DIYbio] Re: Low cost PCR(under development)

On 06/30/2014 06:05 AM, Cathal Garvey wrote:
> t's rare that you'll need decreasing ramp rates faster than 1C, in any
> case. Have you a special experiment in mind?

Are there some good research reasons to go for more delta T rate?
My stirred air cooled PCR cycler concept is likely to do more
when I get around to doing it and testing it. And for evenness,
it will be top performing inherently.

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Very urgent requirement SAS statistical credit risk/risk modelers opening in NY OH

Hello,           

Hope you are doing well,

Please go through the following requirement and let me know on Dhruv@riderconsultinginc.com if you have any available resources

 

 

                                                                               

 

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*      Good understanding of optimization algorithms, working experience is preferred
*      Ideal candidate would have 5+ years experience with minimum of a Master's degree in a field that provides a strong background in finance, economics and statistical methods. Ph.D. degree is preferred.

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Email : Dhruv@riderconsultinginc.com
Gtalk : rider.dhruv1

 

 

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Re: [DIYbio] Re: Low cost PCR(under development)

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There are two ramp rates; ramp while increasing, and ramp while
decreasing. Most machines can achieve 3-4C increasing because they just
use heating coils, but their decreasing temperature (accomplished with a
peltier, cooling fluid, etc.) may be lower.

IIRC, OpenPCR has a climbing ramp rate of 2-3C, and a reducing ramp rate
of 1C. Larger machines with fundamentally the same architecture (coil +
peltier) achieve higher rates merely by adding extra wattage.

Remember though, an additional consideration is how homogenous the
temperature change is through the block, and at what rate the
temperature change in the block is reflected in the tubes. There would
be little point doubling the ramp rate of the block if the tubes cooled
at an only marginally faster rate. Worse, doubling the ramp rate as
measured by the thermometer may look great on the charts and benchmarks,
but the block may be unevenly cooling and this can have real
consequences when you're expecting your samples to experience identical
conditions.

It's rare that you'll need decreasing ramp rates faster than 1C, in any
case. Have you a special experiment in mind?

On 30/06/14 10:59, Michael Shamberger wrote:
> OpenPcr has a ramp rate of 1 degree C/s. I checked specs for some
> commercial versions and they have ramp rates of 3 or 4 degrees C/s. What
> are they doing differently?
>

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Re: [DIYbio] Re: Low cost PCR(under development)

OpenPcr has a ramp rate of 1 degree C/s.  I checked specs for some commercial versions and they have ramp rates of 3 or 4 degrees C/s.   What are they doing differently?

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Re: [DIYbio] Re: Low cost PCR(under development)

hoi zäme,

urs has been actively reverse engineered and improved the openPCR:

a good thermal contact is crucial. just an eppi holder usually doesnt really do the trick, but it's fun to make these kinda Jugaad-PCR machines:

and about bangalore... go and see yashas and the peeps at sristhi/NCBS out in yelahanka town. they are doing bio-stuff since 2008 and have an opportunity to create a larger space now.

all the best,
marc

On Sunday, June 29, 2014 6:18:08 PM UTC+2, John Griessen wrote:
On 06/29/2014 10:37 AM, Michael Shamberger wrote:
> I have seen several DIY PCR designs and I was wondering why those are not able to hold standard PCR plates?

Instead of DIY, there could easily be low cost for sale with such if enough line up to buy.
I'll make it.

John Griessen

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Re: [DIYbio] automated fluorescent microscope

Well, with fluorescent imaging, you barely need machine learning as nothing else in the image is visible besides the labelled organisms. 

ie. for identifying normal bacterial colonies, I have to use a dozen or so filters, to correct for background illumination, do complex adaptive thresholding etc etc. For identifying fluorescent bacterial colonies, I just threshold all pixels that are 3x the standard deviation of the image. For bacteria, you could then just do a watershed, and filter on the size of the detected blobs.

As far as finding filters goes, search for "dichroic mirror" on ebay, and select the filter you need from the spectral info. for fluorescent imaging, make sure the excitation and emission bandpass ranges are seperated by at least 15nm to limit bleedthrough. If you are not doing multispectral imaging (eg GFP, YFP and RFP together), then excite at the blue end of the emission spectra, and use a longpass filter at the emission maxima of the fluorescent protein you are imaging. You'll need a dichroic mirror to separate the excitation and emission light too. It's cutoff should be in between the bandpass ranges.

You can look at the spectra and plan using this web tool:
https://www.lifetechnologies.com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

-Dave

On Saturday, June 28, 2014 7:22:17 PM UTC+2, Michael Shamberger wrote:
They are updating the microscope design this summer.  So far they have tracked a nematode.  

>> found the openlabtools data a few months ago 
and ordered what should be an appropriate tube lens for monochromatic 
use.

What did you end up buying?

>> Why do you want to start with fluorescense? Is there anything you 
actually want to see using this?

I want to see if I can track/image live bacteria with a DIY setup costing less the 1K. Then I want to try various machine learning techniques on the images to see if I can reproduce some of the latest research in this area.


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Re: [DIYbio] Re: Low cost PCR(under development)

On 06/29/2014 10:37 AM, Michael Shamberger wrote:
> I have seen several DIY PCR designs and I was wondering why those are not able to hold standard PCR plates?

Instead of DIY, there could easily be low cost for sale with such if enough line up to buy.
I'll make it.

John Griessen

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Re: [DIYbio] Re: Low cost PCR(under development)

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Because nobody who made one wanted high throughput. If you do, then why
not make a project of it, and adapt one to handle plates?

On 29/06/14 16:37, Michael Shamberger wrote:
> I have seen several DIY PCR designs and I was wondering why those are not
> able to hold standard PCR plates?
>
> For example, there are 24,48,96 plates available that are a few dollars
> each and certified sterile.
>

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[DIYbio] Re: Low cost PCR(under development)

I have seen several DIY PCR designs and I was wondering why those are not able to hold standard PCR plates? 

For example, there are 24,48,96 plates available that are a few dollars each and certified sterile.  

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[DIYbio] Re: Low cost PCR(under development)

Enrico,
                 It will be different if we are using a bulb.But it can be solved by using a heating bed(used n 3d printers).

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