Re: [DIYbio] Re: 4-tube incubator and optical density meter arduino shield


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On Wed, Jun 28, 2017 at 5:34 PM, Evgeny Vlasov <vlasov.ca@gmail.com> wrote:
Hi!
That is one very interesting link, exactly what I was looking about. Thank you so much.
Link itself is broken but .pdf is searchable. Tomorrow I am going to nag lab's bacteria guy with this project. 

Cheers

Evgeny


On Thursday, October 7, 2010 at 4:34:43 PM UTC-6, ByoWired wrote:


On Oct 7, 5:46 pm, Mackenzie Cowell <m...@diybio.org> wrote:
> I'd like to build a simple incubator+OD meter for up to 4 microcentrifuge
> tubes....

My first quick comment is that you might have a look at the following:

http://soslab.ee.washington.edu/mw/images/e/ef/EE449_SP10_Group6_MS5_Report.pdf

In that, they used LEDs and a Light-to-Frequency Converter called a
TSL230 which I think they interfaced with the Arduino.  They seem
confident in their results.
I'm in the process now of testing an OD system using a similar set-up,
but it's taking OD readings and some other optical properties through
a syringe, not microcentrifuge tubes.  Also, I'm not familiar with the
Arduino and have built my system around the Propeller microcontroller
- sorry.  I have an incubator built around the syringes using non-
inductive resistors, but I haven't yet tested the incubator portion
yet.  I have digital thermometers using DS18B20's.  Although I've
heard of people using PCB traces for heating, it seems to me it might
be easier to use resistors because they can be positioned around the
microtubes without dramas.

Anyway that's it for now.
hope that helps get you started,
Mark

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[DIYbio] Re: 4-tube incubator and optical density meter arduino shield

Hi!
That is one very interesting link, exactly what I was looking about. Thank you so much.
Link itself is broken but .pdf is searchable. Tomorrow I am going to nag lab's bacteria guy with this project. 

Cheers

Evgeny


On Thursday, October 7, 2010 at 4:34:43 PM UTC-6, ByoWired wrote:


On Oct 7, 5:46 pm, Mackenzie Cowell <m...@diybio.org> wrote:
> I'd like to build a simple incubator+OD meter for up to 4 microcentrifuge
> tubes....

My first quick comment is that you might have a look at the following:

http://soslab.ee.washington.edu/mw/images/e/ef/EE449_SP10_Group6_MS5_Report.pdf

In that, they used LEDs and a Light-to-Frequency Converter called a
TSL230 which I think they interfaced with the Arduino.  They seem
confident in their results.
I'm in the process now of testing an OD system using a similar set-up,
but it's taking OD readings and some other optical properties through
a syringe, not microcentrifuge tubes.  Also, I'm not familiar with the
Arduino and have built my system around the Propeller microcontroller
- sorry.  I have an incubator built around the syringes using non-
inductive resistors, but I haven't yet tested the incubator portion
yet.  I have digital thermometers using DS18B20's.  Although I've
heard of people using PCB traces for heating, it seems to me it might
be easier to use resistors because they can be positioned around the
microtubes without dramas.

Anyway that's it for now.
hope that helps get you started,
Mark

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[DIYbio] Re: I'm a beginner and interested in learning Biohacking but don't know how to and where to

https://www.youtube.com/watch?v=Ds8ZFzOwGeI

Try replicating the Thought Emporium's project on the Scoby Project that's the place I started but that's because I have some lab expierence in my belt as a Biology major.

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Re: [DIYbio] Paralens fluorescence microscope objective for Hoechst and DAPI?

Hey,

DAPI/Hoechst excitation require UV light, so if there's a UV filter in front of your camera, you'll see only the fluorescence signal.
However, common glass is not transparent to UV, so it might be that if you're using cheap glassware in the beam path you'll have a problem.

The 100X objective does not let much light go through it, but Hoechst and DAPI have a very high signal-to-noise ratio, they're very bright, so even in an hacked system there shouldn't be much of a problem.

You can find microscope cameras around, but does your microscope allow for a camera? Is it a trinocular? You can spend additional money for a camera and a C-Mount and hack light source and filters, but probably at that point the cost becomes too high.
If you wanna go DIY, then I would hack the system completely. You can just stick a raspberry Pi camera in front of the ocular (possibly with a 20X magnification if you have it) and it should come with a UV filter (I don't know how much useful). 

There are UV leds that you could use for illumination, you have to make sure that they would provide enough light.
Filters in your case is easy, you can find UV filters.

The solution with the UV light source in one ocular makes sense and it's probably the only one available to you (beside the paralens): you need the light to point away from your eyes and have the fluorescence reflected back, differently to what you are probably used to in brightfield.

To get more info on how to setup something like this, you can have a look at this paper here: http://biorxiv.org/content/biorxiv/early/2017/03/31/122812.full.pdf

If you're going to experiment, please keep in mind that you don't want UV light in your eyes. Be careful.

Best,

ukitel

On Saturday, 24 June 2017 09:36:52 UTC+2, Mike wrote:
Abizar,

Thank you - yes I'd be very interested in trying the setup you suggest - please can you post an image? I have Zeiss Standard 25 binocular microscope. The key thing is that I need a sharp fluorescence image with a 100x oil objective.

- Is the setup you suggest compatible with this type of objective?
- Where can I get the required parts and how do I make them fit my microscope (LED, filters, camera)?
- Can I acquire LED lighting that will provide for UV illumination (for use with DAPI, Hoechst)?

Roninlaw,

The price of the core Paralens Advance system with a single objective lens type is approximately £900 including VAT.

Thanks guys,

Mike

On Friday, June 23, 2017 at 11:15:21 PM UTC+1, Roninlaw wrote:
Price?

On Fri, Jun 23, 2017 at 2:25 PM, Mike <alanmb...@gmail.com> wrote:
Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

(Please see page 9 of the PDF for an illustration of light spectra)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). [b]Question[/b]: Will this device work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike

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Re: [DIYbio] Paralens fluorescence microscope objective for Hoechst and DAPI?

Abizar,

Thank you - yes I'd be very interested in trying the setup you suggest - please can you post an image? I have Zeiss Standard 25 binocular microscope. The key thing is that I need a sharp fluorescence image with a 100x oil objective.

- Is the setup you suggest compatible with this type of objective?
- Where can I get the required parts and how do I make them fit my microscope (LED, filters, camera)?
- Can I acquire LED lighting that will provide for UV illumination (for use with DAPI, Hoechst)?

Roninlaw,

The price of the core Paralens Advance system with a single objective lens type is approximately £900 including VAT.

Thanks guys,

Mike

On Friday, June 23, 2017 at 11:15:21 PM UTC+1, Roninlaw wrote:
Price?

On Fri, Jun 23, 2017 at 2:25 PM, Mike <alanmb...@gmail.com> wrote:
Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

(Please see page 9 of the PDF for an illustration of light spectra)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). [b]Question[/b]: Will this device work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike

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Re: [DIYbio] Paralens fluorescence microscope objective for Hoechst and DAPI?

An alternative is to place your illuminating led and filter on one eyepiece of a binocular head. And a filter and camera on the other eyepiece. The led beam is semi focused on the specimen through the objective. Fluorescence light goes up the optical path and is divided equally into the eyepiece though only the camera sees the fluorescence image.
Can send you an image of this setup if it helps.

On Jun 23, 2017 2:25 PM, "Mike" <alanmbeattie@gmail.com> wrote:
Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

(Please see page 9 of the PDF for an illustration of light spectra)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). [b]Question[/b]: Will this device work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike

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Re: [DIYbio] Paralens fluorescence microscope objective for Hoechst and DAPI?

Price?

On Fri, Jun 23, 2017 at 2:25 PM, Mike <alanmbeattie@gmail.com> wrote:
Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

(Please see page 9 of the PDF for an illustration of light spectra)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). [b]Question[/b]: Will this device work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike

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[DIYbio] Paralens fluorescence microscope objective for DAPI and Hoechst?

Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

http://www.druckerdiagnostics.com/media/wysiwyg/paralens-advance/PLA_Manual_No_Spreads_.pdf
(Please see page 9 of the PDF for an illustration of light spectra)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). [b]Question[/b]: Will this device work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike

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[DIYbio] Paralens fluorescence microscope objective for Hoechst and DAPI?

Hello all,

As a cheap alternative to a fluorescence microscope for my home lab, I am considering the Paralens Advance screw-on objective lens.

http://www.druckerdiagnostics.com/media/wysiwyg/paralens-advance/PLA_Manual_No_Spreads_.pdf
(Please see page 9 of the PDF for an illustration of light spectra)

The Paralens produces excitation at 385-480nm and emission at 480nm (as far as I can tell, please see PDF above!). [b]Question[/b]: Will this device work with Hoechst 33258 and DAPI stain (spectra as below)?

- Hoechst 33258: excitation: 352, emission: 461
- DAPI: excitation: 358, emission: 461

Thank you,

Mike

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Looking for Sr. Python Developer , WA

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Re: [DIYbio] Re: SLICE -- Seamless Ligation Cloning Extract... or, using lambda phage homologous recombination in-vitro

Probably because it requires an extra PCR reaction. 

The separate SLiCE "enzyme mix" you refer to costs nothing, and with about 2 hrs of prep work one can make enough for 10,000+ reactions. PCRs are not free, often require troubleshooting and can be time consuming. The fewer PCRs one has to do the better in my book. 

Granted not all cloning methods work equally well for all situations, but for standard insertion of an insert into a linearized vector it's pretty hard to beat the convenience, cost, efficiency and accuracy of recombinase-expressed cell extract SLICE methods. I suspect those making $$ on Gibson reagents are working hard to ensure these methods don't become commercialized. 



On Wednesday, May 3, 2017 at 11:10:57 PM UTC-7, Michael Crone wrote:
Why not just use CPEC instead of requiring a separate enzyme mix just for cloning? Phusion works absolute wonders and I've never had any problem creating large plasmids.

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Re: [DIYbio] Re: Gibson vs SLiCE (single part recommendations)

Thanks for your input Cihan. I would also be particularly curious to see how the plasmid-based recombinase extract works compared to PPY/genome-based.

The Motohashi papers on using traditional extracts are quite interesting. Thanks for sharing. I've read through his original paper (https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-015-0162-8) and he certainly gets very different numbers for non-recombinase extract than Zhang did, in both cloning efficiency (up to 2-orders of magnitude better) and ideal homology overlap length (20bp compared to 50bp from Zhang). Reasons for the discrepancy are not well discussed. The reported cloning accuracy when combining two inserts into a vector was kind of low as well. In our lab now with the PPY strain we are putting in 1-4 fragments at a time with nearly 100% accuracy, and with good enough efficiency to assemble large libraries. 

This is not to say the methods of using traditional cell extracts don't work well or aren't perfectly suitable for many labs and many applications...I suppose my point is that I am a believer at this point that making extracts from cells expressing recombinase (whether PPY or plasmid based) really does make a difference. And when students clone more efficiently, projects move forward faster. 

One of these days I'll get around to doing a more systematic side-by-side comparison of using extracts with and without recombinase.

Just my two cents. ;)




On Tuesday, June 20, 2017 at 1:13:07 AM UTC-7, Cihan AYDIN wrote:
I also got an MTA for the PPY strain but the shipping costs (for cold chain) was too much for me so I opted for getting a pKD46-deriven plasmid (pREDIA from Addgene). The only difference is in PPY gam and redb is integrated into the DH10B genome and reda in plasmid whereas in pKD 46 it is on the plasmid. I really would like to make a side-by-side comparison for the PPY and pKD46/DH10B in my lab.

Also there is this japanese guy Motohashi who does SLiCe from regular bacteria extracts (recA-) and has data on its efficiency with 15-19 bp overlaps between parts (https://link.springer.com/protocol/10.1007%2F978-1-4939-6472-7_23). He is using JM109 as his primary strain and uses a few more alternatives as far as i remember.

On the note, Gibson is too pricey for any lab who's on a budget, especially DIY labs and labs in developing countries. SLiCe and even CPEC has become better alternatives for synthetic biology and even simple cloning reactions - basically your only cost is bacterial reagents and primers (and PCR reagents in some cases).

On a last note, also read this article - www.ncbi.nlm.nih.gov/pubmed/26463009 - where they combine CRISPR with Lambda phage recombination system to efficiently modify e. coli genome.

Cheers!

C

On Sunday, June 18, 2017 at 6:07:48 AM UTC+3, Rob C wrote:
Not too long ago we obtained the PPY strain as a kind gift from the original makers (Zhang at Albert Einstein). 

In our hands making extracts from this strain works WAY better than traditional DH10b, DH5alpha, etc (using the CelLytic lysis B reagent...it's cheap for what you get). 

Although I don't have exact numbers to report, we have done several tests side by side with Gibson, and PPY based SLICE gives at least equal transformation efficiency. Accuracy is equally as good, and in some isolated cases it has been better. 

After several month of heavy cloning, our lab has now dropped purchase of all Gibson reagents. 

I'm convinced that if more people implement the PPY-based SLICE cloning in their lab, Gibson sales will go out of business. 



On Thursday, March 16, 2017 at 12:32:35 PM UTC-7, Rob C wrote:
Bryan, thank you very much for sharing your experiences. Sounds like you did all the right controls to get a proper assessment of the system. Logically it is a little surprising that there's no carry over of plasmid, but of course very good news and just goes to show the best way to answer a question is to just do the experiment. I tend to see too many people over thinking things to a point where they will convince themselves something is not feasible before they've even tried it, so the project stops before it even begins. :)

Cheers.

On Monday, March 13, 2017 at 11:57:49 AM UTC-7, Bryan Jones wrote:
I would imagine there is some risk of that, but I haven't seen that happen in my experiments. I carried out a negative control using just SLiCE extract with competent cells (No insert DNA), and didn't get any colonies on amp plates. Also the colonies I sequenced from my tests were all correct, no pKD46 carryover. It might be that DNAse breaks all the DNA from the SLiCE cells. Normally when you do a plasmid prep, you add EDTA to prevent this, but no EDTA was added here. I'm not positive why, but empirically, I can say that it does not seem to be an issue. 

On Tue, Mar 7, 2017 at 6:16 PM RBC <rco...@gmail.com> wrote:
Is there any worry that the amp resistant pKD46 plasmid will be extracted into the SLiCE lysate, in which case this plasmid will carry through to the final transformation? To me it would seem you can't use it to make amp resistant vectors..or maybe I'm missing something. Thanks for the clarification.

RC


On Friday, October 21, 2016 at 11:22:11 AM UTC-7, Bryan Jones wrote:
In my most recent test, without pKD46, I got 10 colonies from the SLiCE reaction, which was the same as the no SLiCE control. SLiCE with pKD46 resulted in 50-60 colonies. For comparison, NEBuilder resulted in about 100.



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