Re: [DIYbio] Free Taq

First, ROX is used as a passive reference in RT-PCR to normalize results. Many, although not all, machines use a lamp to excite fluorescence. Since the excitation is not uniform across the plate, the ROX is used to normalize the results, thus providing well-to-well consistency. It also normalizes for volume variations. So, in essence, ROX (a rhodamine dye) is nothing more than an internal standard.

Next, expiration dates. This has always been a topic of contention with me. Life puts expiration dates on everything and the dates seem to be shorter and shorter. One thing they put expiration dates on is sequencing polymer. Question, why would acrylamide ever go bad? But Life puts a three month expiration on a bottle? The answer is pretty simple, if they put an expiration on the bottle we will throw it out AND BUY MORE! Its marketing. Oligos don't expire, polymer doesn't expire, many of the products we consume don't expire..but there is still an expiration date, why? Well, some of the products we use do expire, for instance enzymes loose activity, so companies take advantage of this fact and put expiration dates on everything; SO WE BUY MORE!

My recomendation, use common sense. Do your own stability study. We're scientists and should know how to measure change as a function of time for the products we consume.


Regards;



J

J Adams
Azco Biotech, Inc.
3626 Ocean Ranch Blvd.
Oceanside, CA 92056
t. 858-525-2770

incisive systematics <matt@insysx.com> wrote:

>Hi Guys,
>
>I come from the marketing side of the biotech business. Currently I'm a
>biotech marketing consultant (http://www.insysx.com) after working for
>companies such as Invitrogen, BioTrove (Life Tech) and Affymetrix.
>
>So time to give you the definitive answers to Jonathan's and Nathan's
>speculations above.....
>
>First QuantaBio used to be the manufacturer of BioRad's real time PCR mixes
>... including for the BioRad iQ machines. Hence the mix which is specific
>to that machine. Other manufacturers' machines (e.g. ABI, Qiagen) generally
>require a passive reference dye called ROX to even out volume differences
>from well to well in the PCR plate. So that's the ROX ingredient in the ROX
>mix. Low ROX is a particular low concentration ROX variant variant on that
>system for reasons i won't get into here.
>
>The CT in PerfeCTa is actually really cheesy marketing (says the
>marketer!). In real time PCR / qPCR analysis the key measure is the PCR
>cycle where fluorescence of the amplified DNA exceeds the threshold
>(background). This is referred to as the Threshold Cycle or CT. In
>quantitative real time PCR, s difference of 3.3 cycles in CT represents a
>ten fold difference in the quantity of original target. E.g. if one sample
>has a CT of 10 and another sample has a CT of 13.3, the first sample has a
>ten fold higher abundance of the target.
>
>Best,
>
>Matt Lawes PhD MBA
>Principal
>InSysX LLC
>
>On Friday, August 24, 2012 2:29:10 AM UTC-4, Nathan McCorkle wrote:
>>
>> On Thu, Aug 23, 2012 at 10:16 PM, Jonathan Cline <jnc...@gmail.com<javascript:>>
>> wrote:
>> > Is it just me or does anyone else have trouble keeping up with these
>> crazy
>> > marketing names like
>> > PerfeCTa® SYBR® Green SuperMix for iQ™"
>> > versus
>> >
>> > PerfeCTa® SYBR® Green SuperMix, Low ROX™
>> >
>> >
>> > (rolls eyes)
>> >
>> > They might as well assume I'm a biologist or something, ha. It's a part
>> > time job just keeping up with the latest kit names.. maybe even worse
>> than
>> > comparing Cisco network equipment!
>> >
>>
>> well the iQ is a type of qPCR machine, I dunno what ROX is... I
>> imagine the 'CT' in perfeCTa has something to do with them trying to
>> say it binds indiscriminately to GC or TA pairs. That could all just
>> be what they /want/ me to think though...
>>
>> >
>> > Anyway, it will take me 6 mos to a year to use the samples I've got.
>> Yet
>> > the max shelf life rating is "2 mos at +20C". The oldest I've used is
>> about
>> > 1 year and didn't notice much of a difference, but I have low yields on
>> > experiments anyway. I've only seen one manufacturer publish a curve for
>> > "reliability vs shelf life", otherwise info is ad hoc depending on who's
>> > using what in the lab. Too bad this data isn't part of the normal
>> product
>> > spec.
>> >
>>
>> Also,
>> From here:
>>
>> http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.34900.File.dat/Focus%20Voume%209%20Issue%203.pdf
>>
>> "Making effective use of cloned M-MLV reverse transcriptase"
>>
>> "In addition, keeping three of the cloned M-MLV RT preparations which
>> were 10 to 24 months old at 4 C for 7 days had no apparent adverse
>> effect upon the ability of the preparations to synthesize 7.5 and 9.5
>> kb cDNA from the RNA Ladder (data not shown). These results indicate
>> that purified cloned M-MLV RT has a shelf-life at -20 C in excess of 2
>> years and is not adversely affected by exposure to 4 C during shipping
>> or manipulations in the laboratory"
>>
>>
>> >
>> >
>> > ## Jonathan Cline
>> > ## jcl...@ieee.org <javascript:>
>> > ## Mobile: +1-805-617-0223
>> > ########################
>> >
>> >
>> > --
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>> >
>>
>>
>>
>> --
>> Nathan McCorkle
>> Rochester Institute of Technology
>> College of Science, Biotechnology/Bioinformatics
>>
>
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