Re: [DIYbio] Re: polymerase expr/purification WAS:Anyone need some synthesis?

Unless one is amplifying >1.5kb Taq should not be an issue. Though people remark on Taq polymerases "high error rate" when cloning this is often not an issue because at the end of the experiment one is only using a single copy of the gene to insert into a plasmid. Sequence two clones and as long as you did everything correct your chances should be very high that one or both of them contain the correct sequence.

His-tagged polymerases are very easy to purify if you have access to Nickel.Metal affinity column. Many labs purify their own Taq for things like genotyping that require sooo much polymerase, hundreds of reactions a week. But otherwise it is like trying to make a cheaper hamburger than McDonalds, you will not come close to price matching unless you purchase in large bulk.

To me time is money and to most labs time is money and purification takes time and effort and usually there is no QA involved.

Thermo sells Phusion for $82/100 rxns which is a master mix
http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/phusion-flash-high-fidelity-pcr-master-mix/

Buying dNTPs for ~200 rxns is $30 alone
http://www.thermoscientificbio.com/general-reagents-and-accessories/dntp-mixes/


So let's say one liter of bacteria gives you enough protein at the end for 1000 rxns which would be $820
Gene synthesis: let's say it's only around $200 for arguments sake(pfu is ~900AA? so thats >2000bp more like thousands of $$)
Cloning? (Restriction Enzymes, &c.) Add what you think is reasonable
~$100-150 is the cost of the dNTPs for 1000rxns
Let's say reagents and stuff for expression.lysis.storage $150
A day or two or three or seven or 20 of work optimizing the protocol so your expression and purification work ~$150(let's keep it cheap because this is DIY) the verifying that the polymerase works figuring out how much polymerase needs to be added for each batch, verifying the purity of your enzyme, removing an DNAse contamination, &c.
Nickel or Ion exchange column($50-$100 probably much more but let's keep it cheap)

Your cost is at least $700 even if it was only $400 or $300. Think about that you won't begin to make your money back until you run close to 1000 rxns and even then it's not like you are saving $$$$ you are only saving $. And most likely your cost is going to be in the couple thousand dollar range

For $400 USD you can run about 500 PCR reactions. If you are running 500 PCR reactions either something is wrong and your protocols are failing or you are using consumables at such a high rate that you need to find $$$$ in funding. I think for my whole Ph.D. thesis I ran about 500-1000 PCR reactions over 5 years and cloned 200-400 constructs.

As Sebastian said enzymes are cheap. And they will probably only become cheaper. But I am sure other people feel different.



Josiah



On Friday, March 21, 2014 5:51:33 PM UTC-7, Koeng wrote:

Yep that was the idea. Error correction. A lot of the time I need to do HUGE PCRs which taq is not good at (So many errors) in which case I use Q5. 

By the way, anyone know what Q5 polymerase is? Its like over 100x less error prone then Taq and like 3x as fast. But I couldn't find an organism they got it from... and when I looked at their patents last (it was a while ago) it had something on DNA binding domains. Might be a synthetic polymerase

Back on topic, pfu didn't seem to have any and was a decent size, only like 2300bp or something. Intiens... those sound interesting. (It might have some relevance to another project I am doing, thanks for bringing it up!)
On Friday, March 21, 2014 5:33:27 PM UTC-7, Cathal Garvey wrote:

Taq's error rate is an issue, but adding a small amount of proofreading
polymerase can make up for that. So you taq's speed and affordability,
and error-correction/processiveness provided by other pols.

I think the apparent difficulty in the early papers can probably be
heavily attributed to poorly optimised DNA; Thermus aquaticus is hardly
a close relative of E.coli. I haven't looked at the codon usage tables,
but I wouldn't be surprised to see an issue there.

Same goes for most thermophiles, so I'd caution against using them
without even a little optimisation? :)

Also, watch out for inteins, I recall finding a proofreading thermopol
in uniprot that was *huge* but a large portion of that was an intein, so
could be dispensed with when designing an artificial gene if you
identify the intein splice sites and chop everything between them out.

That also introduced me to the existence of inteins, which are bonkers.

On 22/03/14 00:24, Sebastian Cocioba wrote:
>   I know u hate taq but did u see the eBay offer of $23/1000u? And I
> believe 50,000u for $200. Thats cheap. SydLabs has a one time deal of 20%
> off an already cheap price too.
>
> Sebastian S. Cocioba
> CEO & Founder
> New York Botanics, LLC
> Plant Biotech R&D
>  ------------------------------
> From: Koeng <koen...@gmail.com>
> Sent: 3/21/2014 8:19 PM
> To: diy...@googlegroups.com
> Subject: [DIYbio] Re: polymerase expr/purification WAS:Anyone need some
> synthesis?
>
> It was just my idea to do pfu because look at how cheap they can make
> taq... it would be essentially the same protocol
>
> and NEB's stuff is like a dollar a PCR
>
> On Friday, March 21, 2014 3:39:09 PM UTC-7, phillyj wrote:
>>
>> On Fri, Mar 21, 2014 at 5:25 PM, Koeng <koen...@gmail.com <javascript:>>
>> wrote:
>>>
>>> Hey
>>>
>>> I am doing a large synthesis order soon from gen9. However, they insist
>> upon synthesis of at least 25kbs, and I unfortunately only need 20kbps
>> synthesized. If anyone wants to get some genes for cheap, contact me. It is
>> 20 cents a base pair ( .5 to 3kbp) and as a little incentive I will also
>> include a (native) pfu polymerase gene for expression in E coli :)
>>>
>>
>> How hard is it to express and purify polymerase in E coli? I was
>> browsing Engelke's paper on it. It looks complicated and not easily
>> done unless you got some specific equipment. Otherwise, it's very
>> doable. Anyone here tried this?
>>
>> Engelke, David R., et al. "Purification of Thermus aquaticus DNA
>> polymerase expressed in Escherichia coli." Analytical Biochemistry
>> 191.2 (1990): 396-400.
>> Available at:
>> http://deepblue.lib.umich.edu/bitstream/handle/2027.42/28292/0000046.pdf
>>
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