first, cheap and effective termal cycler PCR at home

[NicVied]

Hello everyone,

I am biology teacher and I discovered this DIYbio movement one week ago, and it is amaizing. I am thinking of buying a thermal cycler for me and maybe then bring it somehow to the school.

I would like to know your opinion about the 2 opensource thermal cycler that I have found:

https://shop.tori.st/product/ninjapcr-kit/

I have read about this one and it seems it works well.

http://www.gaudi.ch/GaudiLabs/?page_id=873

This is much cheaper and for me would be perfect buy this one, but I know nothing about this one and it seems too simple...

Futher, I would like to ask if you  think it is a bit crazy and if it's going to be too much
expensive the rest of material, primers, buffers,... If I just want to play in my home.

thanks,

[Dakota Hamill]

Can't recommend one since I've never used any of them but there's also.

https://openpcr.org/

https://www.minipcr.com/

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[Toni Nicolau Viedma]

Thanks! But openPCR just has qPCR and it is too expensive. The simple one you can't buy it anymore

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[Avery louie]

haha @Dakota Hamill it has been a while.

@NicVied, welcome! I think the real question is what you want to do with it.  There are a few features to look for when picking a pcr machine:

1) heated lid- without a heated lid (pocketpcr does not have one) you will need to add mineral oil to your samples so that they dont evaporate and condense all over the tube.  not a huge pain if you are doing large or rather infrequent samples, but it will be a pain if your samples are tiny and you are doing a lot of them.  The other one appears to have a heated lid.

2) number of samples.  if you are doing some big science and you want to also run a negative or positive control to help troubleshoot your reaction, you will run out of space in the pocket pcr pretty quickly

3) thermal design- for the open source ones, look at what temperature sensor is in there and how accurate it is.  Does the layout of the heater make sense?  For tricky reactions, you might need to command the temperature very precisely.  This is certainly not a slam vs DIY machines but its something to keep in mind.  That said, temperature control is not always super critical.  One time we made one (Dakota may have been there) with a lightbulb and a fan.  And it worked.

4) consider a used commercial machine.  usually the interface is a little clunky but you are getting a product that has actually been tested as lab equipment and probably has a UL or CE cert, that you don't have to build and troubleshoot yourself etc.  Usually these have nice enclosures and can cost the same as a kit.  You can pick one up (or a whole lab) from the odin (I think).

Fortunately, a lot of these machines, in particular openpcr and minipcr have been around since about the beginning of diybio, so they are a lot better than they were 10 years ago, and they are about a million years ahead of commercial machines in terms of UI and hackability (at least vs the commercial machines I have seen).

Good luck!  let us know what you end up buying.

--A

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[jlund256]

PCR was originally done by setting up three water baths at different temperatures (annealing 55-65C, extension 72C,  denaturation-boiling), and moving the PCR reaction tubes by hand between water baths.  This is the easiest way to test PCR.  Estimate about 2 min total per cycle, in one hour a PCR amplification can be done.  This gets tedious--sitting there, watching a timer--which is why PCR machines were developed, but you can start trying it without building/buying a machine.

Cheers,

Jim Lund

[Bryan Jones]

I did most of my PhD thesis work using an OpenPCR, too bad they aren't selling it anymore. The Ninja actually looks pretty similar to the OpenPCR (maybe the same heatblock?). If you are close to a research university, I'd also recommend seeing if they have a used/recycled equipment store. Many universities do, and they will often have old lab equipment. I've picked up one or two old PCR machines at the University of Minnesota's ReUse store.

--Bryan Jones

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[Jacob Fooks]

It depends to some extent on how "diy" you want to be and what your skill set is... I cobbled together a serviceable thermocycler that can do a half dozen epi tubes from the cooling block from an old HP workstation I had in my attic, a cartridge heater, an arduino w/ thermocouple, and some other electronic bits and pieces I had lying around. Material cost was basically nothing, but for the time I spent tinkering to make it serviceable I could have picked up a consulting gig and made enough money to buy a nice, shiny, new one...

[NicVied]

Thanks you all, finally I bought the pocket PCR. It's really cheap but without "hot lid", old school style. I will let you know how it work!

[Tom De Medts]

Hi NicVied,

Can you please share a link to this pocker PCR, thank you.

Are you going to later mineral oil in lieu of a heated lid?

If yes, then will you buy it from a scientific lab supplier or just from some marketplace like amazon?

I found this link for the least expensive mineral oil - https://www.amazon.com/Mineral-Pure-Organic-Ingredients-Utensils/dp/B07G5KVW4G/ref=sr_1_33?dchild=1&keywords=mineral+oil+PCR&qid=1599277973&sr=8-33

but I am not at all sure if this will serve the purpose...

I look forward to your response.

Cheers!
TdM

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[NicVied]

About the mineral oil that you find, I would say that could be a problema the DNase, RNase and protease present in it.

Check uct profile of the mineral oil PCR reagent: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/2/m8662dat.pdf

it says:

Product Profile

DNase, RNase and protease: None detected

Suitable for use in the Polymerase Chain Reaction (PCR).

But, maybe if you warm up the mineral oil that you found during 30 minuts at 100ºC the enzymes should denaturalize, don't they?

[NicVied]

Hi Tom De Mets,

Here is the website where I've bought it: http://gaudi.ch/PocketPCR/ We'll see if it works...

About mineral oil, I don't know. I image that the oil should not have rest of DNA, proteases, DNases, or other molecules that can affect the PCR.

Firstly I will try without oil. Somebody in this post said that if your sample is big enough I will not need the oil.

El sábado, 5 de septiembre de 2020 a las 5:57:03 UTC+2, Tom De Medts escribió:

[Dakota Hamill]

I've used $3.99 mineral oil from CVS numerous times and it's never been a problem, 16s and ITS amplifications always work. Just add by eye to form a few millimeter thick layer on top of your sample. 

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[Cathal Garvey]

DNases and RNases are notoriously tough enzymes. :)

But, I think people worry more about these than they should, too. If you use deionised water, then hopefully there are not enough ions around to 'feed' the enzymes before the reaction is prepared and ready to go. Just remember to keep the tubes chilled/on ice before and after the reaction, and consider adding a _small_* amount of EDTA after the reaction to mop up any leftover buffer ions, if you're sure you're finished doing enzymatic things.

Remember though that people were doing molecular work long before the nature of DNases and RNases were even properly figured out. DNases in particular are rarely blamed for bad times, though I know people working with RNA who have blamed RNases for a lot.. but then, you never know. Maybe the real problem was something else and they just assumed RNases were the problem for cargo-culty reasons.

*EDTA is a strong chelator and can be a nasty pollutant if overused, so try to make an effort to use only the amount you need, if you use it at all. Ideally you'd familiarise yourself with molar quantities of whatever ions you're trying to clear up (e.g. Mg++, Ca) and you'd only add a proportional amount of EDTA to the buffer or even less.

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