I haven't used her so far for tricky applications, mostly colony PCR which it works well for. Once I screened 24 liquid culture colonies with it, and it worked out (My cloning didn't though... I got an unheard of 1:16 ratio of correct constructs vs false positives but I finally got a consturct I wanted from the colony PCR. I even got that after gel purification. I now DpnI digest everything)
-- I almost always clone using site directed mutagensis (using NEB turbo cells, I can get a full clone cycle from PCR to transformation to sequencing in 2 days) or gibson assembly, also in a 2 day clone cycle. Unfortunately those require high fidelity polymerase because I can't have the A overhang for it to work as planned. (Which is why I am interested in synthesizing pfu polymerase) Right now I use accura, which works pretty well. I recommend asking for a sample of it. Theres about 20 reactions in the sample, 50µl PCRs.
I'll look into it! I would never use Taq for cloning, but I use it a lot for checking vectors and genotyping, or for short PCRs that I do that I have to use restriction enzymes for.
-Koeng
On Friday, July 25, 2014 9:58:19 AM UTC-7, Sebastian wrote:
On Friday, July 25, 2014 9:58:19 AM UTC-7, Sebastian wrote:
Glad to hear im not the only one crazy enough to buy a 9600! She is built like a tank. Im sure it will outlive me.
How does she fare with tricky or longer amplifications?
Ever run Q5 (98C denat) with it?
I use it for large plant genotyping assays and optimized primers so the machine won't need much precision but never did a cloning run with it for fear of temp drift and all that jazz. Used some of the eBay taq mastermix which I also highly recommend. You can't beat $99 for 2000 10uL reactions! Just search PCR mastermix on eBay. Its sold by Gene and Cell technologies which looks a lot like openbiotech and uses the pOpenTaq vector for production.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Koeng
Sent: 7/25/2014 12:44 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Available PCR machines - comparisons and experiences?
I have one of "the beasts". It works really well, I recommend it, if you can get a good shipping price. I also have another thermocycler which doesn't have a heated lid and is .5ml, that ironically I also use as a dry bath. I got it for a steal because the seller butchered the spelling so it never came up on any searchers (I don't even remember what he put. I just know it didn't come up into search, found it when I was searching for pipettes.) No one had bought it for a lonnnnnng time so I offered 60 dollars with shipping and I got it.--But I think the GeneAmp 9600 is great. Highly recommend for its price!-Koeng
On Friday, July 25, 2014 8:29:58 AM UTC-7, Sebastian wrote:I have the EdvoCycler and its okay. Can't incubate at a fixed temp for more than an hour before thermal runaway sets in and melts my tubes. Learned that the hard way when running a restriction digest for two hours. The machine does work but its nothing in comparison to that gorgeous new PCR vixen from Life Technologies (front page advert will link to it) which costs a little over $4k.
Was toying with the idea of using a peltier, some thermal paste, a thin aluminum sheet, and some custom cast PDMS to make a microfluidic pcr chip that's washable and reusable. Just cast some channels with shrinkydink (enough to hold 10-50uL), leave enough room for thermal expansion, and slap a heat sink and fan behind it. Then just cycle by switching polarity via arduino and temp PID. I know its silly but it may work for routine pcr...
Been looking into the personalPCR like the one Mac sells on his site GeneFoo.com. Im not sure if the price tag matches the parts and labor required. Seems to be marked up like all the others simple due to niche market. Understandable strategy but still to steep for what it is. EdvoCycler is also overpriced. Managed to snag one off eBay for $350. Seller didn't know what he was selling so made it listed "as-is" so I gambled and it paid off. I strongly recommend NOT doing so since more often than not they are sold cheap or acquired cheap due to the fact that they are broken.
More often than not the peltiers are overdriven to meet ramping spec while maintaining a smaller package so they burn out in a few years of use. I spoke to a technician from an osmometer company and he said some of the devices at his company has been utilizing the same peltier device for 20+ years and has not burned out yet.
I've been burned twice on PCR gambles but also lucked out with simple fixes. The other machine I have is the GeneAmp 9600 aka "the beast". Its huge but maintenance is like servicing a car. It runs on prestone antifreeze and uses a minifridge compressor so its an easy fix if broken. It works fine, there are many units on eBay available. Shipping is killer though.
Ask sellers to run diagnostic if interested in any of the Perkins Elmer or applied biosciences devices. The GeneAmp 2400 is the smaller (narrower) variant of the beast and utilizes same coolant technology.
The button sequence is roughly as follows:
UTIL -> DIAG then run the heater test, chiller test, then system diagnostics in that order and ask seller to take a picture of test result screen. Once each test is selected the device will run and the state results with a press any key implied prompt to move to next test. Chiller is the key in this one and if the test hangs for 15min or more then compressor is dead. An audible click should be heard as the relay energizes and then a faint rush as compressor starts to ramp. If temp just drops very slowly then its just cooling due to ambient temp and its a fail.
With these machines its either a lack of coolant (Prestone heavy duty high mileage prediluted) or a dead compressor. The heater rarely ever fails. They all come with a heated lid and its a nice one too.
Anything listed on eBay as an Eppendorf MasterCycler for ~$300 is most likely busted with a dead peltier unit aka DO NOT BUY IT unless diagnostic tests pass. There is a recurring error code in all devices in that price range. IIRC its error 47 which is a power failure with loud annoying beeps but further testing indicates its a peltier block short.
I grabbed a pretty little biometra uno last year for dirt cheap. No heated lid and 0.5mL wells but temp and cooling works so its my precision dry bath for cutting, incubating, ligations, etc. I did run a few test reactions using mineral oil and they worked....just not ideal.
All in all im not sold on the current "Low Cost" devices on the market. OpenPCR is tragic for the price range especially if it's a kit where results may vary greatly. LavaAmp looked great even though its single tube. Light bulb seems neat too and maintains the super low cost ethos. You can find an old, working, PCR machine for $600 on eBay if you are diligent. I see no need to buy a personalPCR-like device until you've exhausted all auction house avenues. Hope this helps anyone wandering the dark and dank alleyways of Ebay's biotech wing alone for the first time. :)
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: jarlemag
Sent: 7/25/2014 6:11 AM
To: diy...@googlegroups.com
Subject: [DIYbio] Available PCR machines - comparisons and experiences?
--In the last few years, several low cost (in relative terms) PCR thermocycler designs have become available for purchase. I only have personal experience with one of these, OpenPCR. However, other alternatives include PersonalPCR (http://personalpcr.com/) and miniPCR (http://minipcr.com/). Does anyone have experience using more than one "low-cost" PCR design? If so, can you tell a little about the differences, advantages and drawbacks of each? As far as I know, OpenPCR is the only one to use a peltier element for cooling. Do you consider this an important feature?I also noticed that Ezequiel Alvarez-Saavedra is on the team of both PersonalPCR and miniPCR. Does anyone know the signficance of this/the relation between these two? Is the design very similar?Lastly: Does anyone know the status of the LavaAmp project? There have been no official updates for a long time. I vaguely remember reading somewhere that it is still under development and a reason being given for the lack of updates, but I can't recall the details. Does anyone know anything?Best regards,Jarle Pahr
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