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

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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