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Re: [DIYbio] Re: Were the DIY CRISPR kits contaminated?

There is one thing that is not really discussed here: the HM68 cells barely grow at37. When you try to grow up at 37 C, you actually select for even minor contaminations.


On 9 Feb 2018 13:49, "'Wolfgang Nellen' via DIYbio" <diybio@googlegroups.com> wrote:

Hi Josiah,
I largely agree with Rüdiger and Marco.

A refund does not help a lot. It would just be nice if critical reviews of the kit were also published on your website and allow to talk about problems. Our comments on the kit (Science Bridge, Jörg Klug) were never released. (This was a kit ordered independently from the two by LGL, Rüdiger and others). This could have started the discussion a lot earlier prevented a lot of damage to the community.

Wolfgang



Am Montag, 11. Dezember 2017 17:55:16 UTC+1 schrieb ukitel:

Were the DIY CRISPR kits contaminated?


Dear all,


We would like to give you an update on the situation regarding the possible contamination of the DIY Bacterial Gene Engineering CRIPSR kit distributed by The Odin online shop. We believe it is an issue that should be openly discussed as it can potentially affect the DIY biology community as a whole.


In March 2017, the authorities for health and food safety (LGL) of Bavaria, in Germany, issued a press release revealing their results of an investigation on the DIY Bacterial Gene Engineering CRIPSR kit supplied by The Odin, a company selling biotechnology reagents over the web. The LGL stated that the bacterial strain supplied in the kit was contaminated with potentially pathogenic species, such as Enterobacter species and Klebsiella pneumoniae. Based on these findings, the kit was subsequently banned from being used in Germany.


The press release came shortly after Rüdiger Trojok organized an event entitled "CRISPR kitchen". LGL representatives requested the organizers from the Institute for Technology Assessment of Karlsruhe (KIT) to hand out a vial of the bacteria obtained from an Odin CRISPR kit, there on display. At the time, the LGL did not specify that they had already found a contamination in another sample.


After the press release, some people involved in activities of the DIYbio community of Europe felt that a proactive attitude was needed to address the issue in the most transparent manner and organized a volunteer committee.


It has been difficult to gather information from both the LGL and the Odin. The LGL press release did not contain any detailed information and we had only access to a leaked report containing a summary of their results. We managed to get some additional information about the methodology used from LGL, but we could not access any of the raw data. LGL researchers told us they would not release the data until their investigation is completed, and will only publish their results in a peer-reviewed journal at a later point.


The Odin produced blast mapping of the E.coli 16S sequence to their bacterial stock and showing 99% identity. They also showed that the strain was negative for lactose fermentation. Both these results were in contradiction with the findings described in the LGL report.

It was decided that gathering more information was necessary to evaluate the situation independently and start an open discussion on how we can promote safe and lawful use of these and other kits offered in and to the DIYbio community. The goal was to evaluate whether contamination was indeed a systematic problem or an isolated accident.


We gathered 3 samples independently from LGL and analyzed them. Two of these samples came from the incriminated CRISPR kit and the last one was an Escherichia coli HME63 strain identical to that included in the kits, all of them being sold by The Odin and shipped to European customers. To obtain high-quality DNA sequencing data, we secured a collaboration to sequence the samples at the European Molecular Biology Laboratory (EMBL) Genomics Core Facility in Heidelberg, Germany. We discussed with the authorities in Berlin (LAGESO, the LGL equivalent) how to safely handle the samples and shipped them to EMBL for analysis.


EMBL Genomics Core Facility collaborators were able to revive only one of the three samples, which showed bacterial growth both in liquid culture and on agar plate. From the 16 colonies grown on the agar plate, 10 colonies were chosen for library preparation and DNA sequencing on an Illumina platform (MiSeq). The sequencing reads have been uploaded at ENA database under the following ID PRJEB23486. We encourage everyone to go and analyze the data if interested. The Core Facility is analyzing the data, but we wanted to share with you some preliminary results:


The E.coli MG1655 strain is the closest strain relative to the E.coli HME63 supplied with the kit and whose complete genome is available. Therefore, we first aligned the sequencing reads to the E.coli MG1655 genome, but noticed that only 30% of the reads are mapping, which wouldn't be expected if the colonies contained E.coli HME63.

 

We then proceeded to assemble the unmapped reads in large sequences (>50 kb) and checked to which bacteria species they were matching. The analysis returned various Enterobacter species (aerogenes, cloacae and hormaechei), as well as Escherichia coli and Salmonella enterica. However, given the relative phylogenic proximity of these species, with this approach it was not possible to pin down the exact bacteria in the colonies. Nevertheless, it gave an indication of the possible bacteria identity.

 

Given the recurrence of Enterobacter species in the results, we aligned the reads this time to the genome of Enterobacter hormaechei subsp. Steigerwaltii and we could now see that 83% of the reads were mapping and corresponded to an expected good mapping rate on a specific genome. Notably, the presence of Enterobacter species was also mentioned in the LGL report.

 

Moreover, as The Odin made available to us 16S sequence of the E.coli bacteria stock in their possession, we sought to determine if that sequence could be found in our reads. Unfortunately, the reads encompassing the 16S region showed again a much better match to Enterobacter hormaechei rather than E.coli.

 

This suggests that the bacteria in the colonies are most probably an Enterobacter species and not E.coli HME63. The 30% of reads mapping to E.coli are probably the result of cross mapping reads between E.coli and Enterobacter genomes.

 

We would like to mention a few words of caution. First, the methodology we used to obtain these results (whole genome sequencing) is different from the one employed by LGL (metabolic and phenotypic analyses). Second, the sample we analyzed was also different from theirs and a single occurrence could again be anecdotic. Third, we cannot guarantee that the samples were stored appropriately at all times before we collected them and ran the sequencing analysis. However, the fact still remains: both our results and the LGL investigation seem to confirm a contamination of the E. coli strain provided with the kit, possibly with the same bacterial species.


Even though the European Centre for Disease Prevention and Control (ECDC) has pointed out that the risk of infection using such contaminated kit is low, we believe it is not acceptable to have laypeople, often not skilled in laboratory techniques, as it may happen in DIYbio, unknowingly handling potentially harmful biological materials. We believe it is our responsibility to act responsibly, something that was explicitly stated in the DIYbio code of conduct and thus learn from this incident and inform the whole community about it. A failure to handle biomaterials appropriately is detrimental to the goals of the community to democratize the knowledge and technology of life sciences.


Authorities in the EU have to follow generally stricter rules than other parts of the world, including the US. Genetic engineering is particularly heavily regulated and only very few and specific educational experiments can be done outside of authorized laboratories. However, we want to stress here that our findings are not discussing the meaningfulness of European GMO laws. The problem we are concerned with is the shipping of living biological samples, which could contain unwanted bacteria or even potentially pathogenic ones due to insufficient quality assurance procedures. This problem not only unnecessarily endangers the customers, it also calls into question the ability of the DIYbio kit manufacturers.


We believe the value of educational kits needs to be stressed out and we want to support their safe distribution and use. We would like to start a discussion involving DIYbio practitioners, as well as the kit producers, academics, the authorities and the public, to collaboratively develop guidelines, or if necessary develop appropriate and efficient methods and procedures to guarantee the safety of the kits and their users.


We believe this should be a community effort in the spirit of the values of the DIYbio code of conduct and so this is also a call to all of you to get involved. If you have any questions regarding the analysis, please engage in the open discussion here on the google DIYbio mailing list.


Finally we would like to acknowledge the help provided by the EMBL Genomics Core Facility team, Vladimir Benes, Jonathan Landry and Anja Telzerow. Without their work and supportive attitude towards the community this work would have not been possible.


 

Best Regards,

 

Rüdiger Trojok

Marco R Cosenza

Julian Chollet

Luc Henry

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Re: [DIYbio] Re: Were the DIY CRISPR kits contaminated?

Well, Jörg posted on the comments site of The Odin. Apparently they decided that this was not a good comment.


On 9 Feb 2018 23:19, "Nathan McCorkle" <nmz787@gmail.com> wrote:
On Thu, Feb 8, 2018 at 6:54 PM, 'Wolfgang Nellen' via DIYbio
<diybio@googlegroups.com> wrote:
> Our comments on the kit (Science Bridge, Jörg Klug) were never released.

Sounds like that is your fault. Why didn't you post them online publicly?

In the computer security industry there's a common practice known as
"Responsible disclosure", where you let a company know they messed up
and give them ~6 months to try fixing their issues before going
public.
https://en.wikipedia.org/wiki/Responsible_disclosure

Sounds like it's well past time to release your comments publicly.

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Re: [DIYbio] Re: DIY electroporator

if it's a normal 
4mm electroporation cuvette, 100 ul
On Thursday, February 22, 2018 at 11:00:42 PM UTC+5:30, John Griessen wrote:
On 02/21/2018 10:34 PM, nisha p wrote:
> can anyone tell me about the minimum resistance on load present during electroporation and how much current will be drawn
>
Depends on size, volume.

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Re: [DIYbio] Where do DIY Biologists get things like Crispr or genes

maybe...
see this thread...
https://groups.google.com/forum/#!topic/diybio/PXeoidiWPYA


On Saturday, October 21, 2017 at 4:21:56 PM UTC+2, Hans Wilms wrote:
Yeah, the Odin is just an online store for these kinds of things. They don't sell anything dangerous, so it can be shipped right to your house if you live in the right countries.

On Sat, Oct 21, 2017 at 1:58 AM Skyler Gordon <skg...@gmail.com> wrote:
The cool part about a community like this is that it can be used to find like minded people that will want to trade materials, and you only need a business license / tax registration / location to send it.

Most people just don't have -20 freezers at home to store things like host E.colin strains, plasmids, etc.

-SG
On Fri, Oct 20, 2017 at 4:24 PM 'Lauren-Ashley Duncan' via DIYbio <diy...@googlegroups.com> wrote:
Thanks you guys.
I knew there must be a way! So for Odin you can request specific stuff? I do know about the kit they have but we already do stuff like that in the lab I teach. I'm trying to put together some protocols for other stuff after I assess legalities.
And defintely, I'll be shooting you an email at some point (second poster- sorry can't see your name now) after I have some free time and can start enjoying this hobby (right now having too many late nights in my own lab - rushing to complete an abstract). Thank you for the offer!

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Re: [DIYbio] Re: Screw cap petri dishes?

just saw this discussion...
I also am very concerned about plastic waste from lab work...
we put glass petri dishes in aluminum foil for autoclaving.  helps avoid slips...
I don't see that stainless steel could be very useful, however, unless you know you already have a basically pure culture.  (most of my recent microbio has been more on the citizen science end of things.) . Has anyone used these?
best,
Rachel

On Friday, January 18, 2013 at 5:23:20 AM UTC+1, Xabier Vázquez-Campos wrote:
By the way, why do you want them to be tight seal? To avoid the agar to get dry during long term incubation? storage?
For storage, plastic wrap, parafilm or even scotch tape work fine. If the issue is long term culturing as I do, try to get 3M surgical tape, it has a good permeability for gases and helps to keep the moisture of the plates (parafilm is almost gas impermeable and slows down the growth).

As Gra says, they get quite slippery, not only from the fridge but also from the autoclave (unless it is one with dry cycle).

Also, keep in mind what bugs you will culture. You will need to scrap the agar from the plates to clean them.

El viernes, 18 de enero de 2013 14:41:27 UTC+11, John Griessen escribió:
On 01/17/2013 08:25 PM, Sebastian S. Cocioba wrote:
> Its not so much Sterility as it is waste production. It makes me feel crappy thinking of all the polystyrene waste a few
> experiments pile up. There must be a better way...

Sounds like you'd be happy with both halves made of PP.  The lid and the very short jar.
What if the sealing was done by a thick squishy gasket so that a rough 3DP
jar would do?  Then you might autoclave and reuse it, then promote to sell a high enough
volume to get them make of smooth PP from expensive injection molds.

Or just skip the injection molds and 3DP it with advancing smoothness as that progresses,
and get involved in the fight over 3DP with 3DS and Kickstarter and Formlabs 3D printing patent suit?

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Re: [DIYbio] Re: Were the DIY CRISPR kits contaminated?

oops, the facebook page (and the wiki).
here is one...
https://www.facebook.com/events/527517820982197/
best,
RA

On Tuesday, February 27, 2018 at 10:47:28 PM UTC+1, Rachel Aronoff wrote:
After just catching up on this discussion, in its many interesting permutations, I feel I should just add that your solution, Rüdiger, is not only of great interest but also quite topical, as we are just organising a public event about phage therapy at Hackuarium.  (We had another a while ago, apparently, but that was before my time...)
So, anyway, if there is any chance people might be around Lausanne at the end of next month (28march), you are invited to join us, and we can discuss this all further (possibly around a beer!).  The event is already up in the Hackuarium web page.

Just to get back on topic, I applaud all who contributed to solving this issue of the 'banned odin kit,' and hope to see more collaborative efforts and less hype in the future.  DIT Research is the way forward, imho...  (Of course we are very keen on the diybio.org code of conduct, too!)  It seems to me that those who bundle together transhumanism with biohacking are not generally helping, but that is another story, and I am very new to all this.

I would add that I personally am quite interested in trying the non-cutting crispr to probe sub-nuclear dynamics, so if anyone else has great tools for that, please let me know!
Thank you very much!

best wishes!
Rachel




On Friday, February 9, 2018 at 11:19:54 PM UTC+1, Nathan McCorkle wrote:
On Thu, Feb 8, 2018 at 6:54 PM, 'Wolfgang Nellen' via DIYbio
<diy...@googlegroups.com> wrote:
> Our comments on the kit (Science Bridge, Jörg Klug) were never released.

Sounds like that is your fault. Why didn't you post them online publicly?

In the computer security industry there's a common practice known as
"Responsible disclosure", where you let a company know they messed up
and give them ~6 months to try fixing their issues before going
public.
https://en.wikipedia.org/wiki/Responsible_disclosure

Sounds like it's well past time to release your comments publicly.

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Re: [DIYbio] Re: Were the DIY CRISPR kits contaminated?

After just catching up on this discussion, in its many interesting permutations, I feel I should just add that your solution, Rüdiger, is not only of great interest but also quite topical, as we are just organising a public event about phage therapy at Hackuarium.  (We had another a while ago, apparently, but that was before my time...)
So, anyway, if there is any chance people might be around Lausanne at the end of next month (28march), you are invited to join us, and we can discuss this all further (possibly around a beer!).  The event is already up in the Hackuarium web page.

Just to get back on topic, I applaud all who contributed to solving this issue of the 'banned odin kit,' and hope to see more collaborative efforts and less hype in the future.  DIT Research is the way forward, imho...  (Of course we are very keen on the diybio.org code of conduct, too!)  It seems to me that those who bundle together transhumanism with biohacking are not generally helping, but that is another story, and I am very new to all this.

I would add that I personally am quite interested in trying the non-cutting crispr to probe sub-nuclear dynamics, so if anyone else has great tools for that, please let me know!
Thank you very much!

best wishes!
Rachel




On Friday, February 9, 2018 at 11:19:54 PM UTC+1, Nathan McCorkle wrote:
On Thu, Feb 8, 2018 at 6:54 PM, 'Wolfgang Nellen' via DIYbio
<diy...@googlegroups.com> wrote:
> Our comments on the kit (Science Bridge, Jörg Klug) were never released.

Sounds like that is your fault. Why didn't you post them online publicly?

In the computer security industry there's a common practice known as
"Responsible disclosure", where you let a company know they messed up
and give them ~6 months to try fixing their issues before going
public.
https://en.wikipedia.org/wiki/Responsible_disclosure

Sounds like it's well past time to release your comments publicly.

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New Position-Java Developer || Jersey City, NJ || Locals Only || NO OPT

Dear Partner,

Hope you are doing great!!

Please go through the requirement and let me know if you are having any consultant for this position.

Please share me the profile asap as this is a very hot requirement.

 

Java Developer

Jersey City, NJ

6+ Months

 

We're looking for technology "geeks" who have knowledge of emerging technologies and are willing to deep dive into unfamiliar areas of a system to quickly prototype new solutions. This person should have a Java background and be comfortable developing in multiple technology areas while working on prototype and proof-of-concept assignments across the full-stack. Along with excellent communication skills, he/she should be able to demonstrate working with architects on complex assignment and have an overall software engineering experience of 5-8 years. Primary Skills(required): Java, J2EE, Spring, Oracle, Bootstrap, Microservices, ReactJS/Angular JS. Must be strong in the following areas.

 

1)            Experience in building Oracle cloud-based platforms and/or applications with 3+ years of strong Software development background using server side technologies (java, Spring or similar frameworks)

2)            Experience with Full Stack Frontend/UI Development: React / Angular / Javascript / HTML5 / CSS3 / AJAX / Bootstrap

3)            Familiarity with large data storage & processing technologies. NoSQL, Hadoop, Spark are some of the areas that are nice to have.

4)            Familiar with messaging and integration technologies and components. Experience in developing event and message driven applications

 

 

Myscheduling Skills:

             Java – P2

             Spring – P2

             Oracle – P2

             ReactJS – P2

             AngularJS – P2

 

 

Thanks,

                                                                                                                                                                         

Arpit Arora

Pyramid Consulting, Inc.

Executive-Resourcing

 


Desk Phone: 415.943.9386 E: Arpit.arora@pyramidci.com  Web: www.pyramidci.com

 

cid:image009.jpg@01D02C05.4FFC5560

 

WE FIND HIDDEN TALENT

 

How am I doing ?   My goal is to provide you with excellent service. If you have questions, suggestions, feedback about your experience, or need to escalate an issue, feel free to reach out to my manager via email at Mohammed.Tausheef@pyramidci.com or via phone at 415.943.9384.

 

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Rate 50/hr on C2C || New Position-ETL Developer

Dear Partner,

Hope you are doing great!!

Please go through the requirement and let me know if you are having any consultant for this position.

Please share me the profile asap as this is a very hot requirement.

ETL Developer

San Jose, CA

6+ Months

PRIMARY SKILL: ETL Python/Java
Skill Set/Technologies:
Bachelor's Degree in Computer Science, Management Information Systems or related field or equivalent knowledge.
Mandatory:
Strong Experience in coding in SQL
Experience in data warehousing, data management, ETL processes and data modeling tools
(Pentaho/python/java) at least one strong skill set is mandatory

Responsibilities:
Design & Develop basic and advanced transformation activities such as normalization, cleansing, aggregation, summation, and integration; designs automation processes to control data access, transformation and movement; ensures source system data availability and update accessibility, data integrity, ability to resotre and appropriately handle errors, in a time sensitive manner.
Develop testing strategies, including test script formulation and execution, used to validate the accuracy and results of ETL solutions; participation and contribution in quality assurance walk-throughs of ETL components.
Understanding supported business processes, and engage business areas using advanced/expert understanding of business requirements for translation into feasible process.
Develop and document the systems, processes and logic required to expose the existing data sets in the warehouse to end users for reporting and analysis purposes.
Manage multiple broader efforts that span multiple projects/initiatives. Prioritize and monitor project/task status and risk management/mitigation, by working with appropriate manager on level of effort/estimates, resource assignments, etc.
Adhere to standard SDLC procedures and ensure the creation of appropriate documentation.
Ensure adherence to Change Control and release management procedures, including generation of appropriate packages, documentation, and coordination with other technical areas.

Preferred:
Expert with Python/Java, Pentaho, Tableau
Ability to learn and implement new and/or different techniques.
Self-starter with ability to work independently or as part of a team.


 

Thanks,

                                                                                                                                                                         

Arpit Arora

Pyramid Consulting, Inc.

Executive-Resourcing

 


Desk Phone: 415.943.9386 E: Arpit.arora@pyramidci.com  Web: www.pyramidci.com

 

cid:image009.jpg@01D02C05.4FFC5560

 

WE FIND HIDDEN TALENT

 

How am I doing ?   My goal is to provide you with excellent service. If you have questions, suggestions, feedback about your experience, or need to escalate an issue, feel free to reach out to my manager via email at Mohammed.Tausheef@pyramidci.com or via phone at 415.943.9384.

 

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Re: [DIYbio] Re: Home cloning setup

I use the honeywell air purifier - https://www.amazon.com/Honeywell-50250-S-True-HEPA-Purifier/dp/B00007E7RY/ref=sr_1_5?s=home-garden&ie=UTF8&qid=1519684656&sr=1-5&keywords=Honeywell+air+purifier (this one is larger than the one I have, though I couldn't find the one that I do. Must be discontinued)

I probably need to change it, but I haven't had to thus far. It is a bit better than what I need, since I have a small room. 

Awesome :) will ask to ship some soonish

Koeng

On Monday, February 26, 2018 at 12:08:04 PM UTC-8, Roninlaw wrote:
Hi Koeng,

What kind of air filter do you use? Do you have to change it after a certain amount of time?  We have some competent cells you can use. 

Thanks,

Dan

On Feb 26, 2018, at 11:07 AM, Koeng <koen...@gmail.com> wrote:

Hi Flynn, 

I actually do have a cloning setup at home. My lab is actually specifically made for cloning (I like to figure out how to clone things well while working on the Free Genes project, then implement those things at home). And no, I'm not rich. I rent a single small bedroom, which is both my lab space and my home. 

Incubators are cheap from ebay. ~$100. I put an orbital shaker I got for $50 into it, so now I have both a shaking and stationary incubator. You also don't really need an incubator... bacteria grow just fine at room temperature, albeit for longer times. I often grow my sporulating Bacillus at room temperature so they don't take up room in my incubator. 

I don't actually have any freezer room except a small box for enzymes that I keep in our main freezer that I share with roommates. Unfortunately, that also means I don't really have space to store any food, so I usually keep dry food at home for eating. If I had room, I'd pick up a $200 chest freezer from Home Depot, that could store all my stuff for the foreseeable future. Oh, the glories of having space...

I don't have a -80C freezer. Those things are expensive man! And large. Right now I just use competent cells as I make them. That is getting very annoying, so I plan on paying for storage of my competent cells at a local biohacking lab. @Sebastian has also reported that the cells stay good for a few weeks at -20C, so it is quite possible if I had a chest freezer I could just make new batches every other week. 

For long term storage options, I've found that cells last for about a year at -20c in glycerol. They start doing funky stuff after that, so if you regenerate stocks every 6 months, it should be fine. Or just store plasmid stocks. Another good method is to spin down the cells just like you are about to do a miniprep, then freeze those stocks. If you need the plasmid, go ahead and do a miniprep of that frozen stock. It works well after a couple of months, and I haven't tried a yearlong experiment yet. 

You don't need a hood. Why would you need a hood? You don't even need a bunsen burner! Pick up an air filter from amazon for about $150, and that will be MORE than enough. In addition, when you make LB and the dust gets everywhere, the air filter will make sure your room doesn't get all dusty. It also keeps things nice and sterile, just like a bunsen burner. To spread cells, just keep a bic lighter handy and a small beaker of ethanol (from CVS). Dip your inoculating loop in the ethanol, then just do a quick light to burn that ethanol off. Keeps it sterile. 

I use GoldenGate enzymes. No need for PCR reagents. I have 5 enzymes - T4 Ligase, T4 PNK, SapI, BbsI, and BsaI. About $250 off of NEB. I don't like PCR for several reasons. However, if it fits your fancy you can get those enzymes for a couple hundred more bucks.

I don't need microscopes. Why would I need a microscope? Why would I need a FEW microscopes? Here's a tip - learn what your bacteria smell like. I recall one time I caught contamination of my yeast stocks because I took a whiff of the overnight and it didn't have that familiar baker's-yeast smell. Bacillus subtilis is quite easy because it smells so much like dirty feet. E.coli also has a certain smell. You have a great biochemical sensor built into yourself! Use it!

List:
Enzymes : $250
Incubator + shaker: $150
Centrifuge : $100
PCR Machine: $150
Air filter: $150
Glassware: $100
Pipettes: $150
Disposables: $100
Media: $100
Other expenses (tables, boxes, tube holders, etc etc. Everything else you can think of): $250
---
Total: $1500

That seems expensive, but remember I have a very complete lab that I am quite proud of, and probably spent around the range of $2500 on over the range of the last 5 years (high-throughput is expensive ): ). If you're clever, I bet you can cut costs but many hundreds of dollars, and get a cloning setup to do your specific experiment for about $500. If you need any advice, hit me up. But no, you don't have to be rich. I'm certainly not rich, just dedicated :)

If you're not sure how to get all that stuff, Josiah has made it quite easy at the-odin http://www.the-odin.com/genetic-engineering-home-lab-kit/ 

Otherwise, back to eBay!

Koeng


On Friday, February 23, 2018 at 9:32:46 PM UTC-8, Michael Flynn wrote:
Hi.

Does anyone have a home cloning setup - how are you doing it? Are you rich? You must have an incubator, a -20c and -80C freezer, maybe a hood, digestion enzymes and PCR reagents, and a few microscopes? 

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Re: [DIYbio] Re: Home cloning setup

Hi Koeng,

What kind of air filter do you use? Do you have to change it after a certain amount of time?  We have some competent cells you can use. 

Thanks,

Dan

On Feb 26, 2018, at 11:07 AM, Koeng <koeng101@gmail.com> wrote:

Hi Flynn, 

I actually do have a cloning setup at home. My lab is actually specifically made for cloning (I like to figure out how to clone things well while working on the Free Genes project, then implement those things at home). And no, I'm not rich. I rent a single small bedroom, which is both my lab space and my home. 

Incubators are cheap from ebay. ~$100. I put an orbital shaker I got for $50 into it, so now I have both a shaking and stationary incubator. You also don't really need an incubator... bacteria grow just fine at room temperature, albeit for longer times. I often grow my sporulating Bacillus at room temperature so they don't take up room in my incubator. 

I don't actually have any freezer room except a small box for enzymes that I keep in our main freezer that I share with roommates. Unfortunately, that also means I don't really have space to store any food, so I usually keep dry food at home for eating. If I had room, I'd pick up a $200 chest freezer from Home Depot, that could store all my stuff for the foreseeable future. Oh, the glories of having space...

I don't have a -80C freezer. Those things are expensive man! And large. Right now I just use competent cells as I make them. That is getting very annoying, so I plan on paying for storage of my competent cells at a local biohacking lab. @Sebastian has also reported that the cells stay good for a few weeks at -20C, so it is quite possible if I had a chest freezer I could just make new batches every other week. 

For long term storage options, I've found that cells last for about a year at -20c in glycerol. They start doing funky stuff after that, so if you regenerate stocks every 6 months, it should be fine. Or just store plasmid stocks. Another good method is to spin down the cells just like you are about to do a miniprep, then freeze those stocks. If you need the plasmid, go ahead and do a miniprep of that frozen stock. It works well after a couple of months, and I haven't tried a yearlong experiment yet. 

You don't need a hood. Why would you need a hood? You don't even need a bunsen burner! Pick up an air filter from amazon for about $150, and that will be MORE than enough. In addition, when you make LB and the dust gets everywhere, the air filter will make sure your room doesn't get all dusty. It also keeps things nice and sterile, just like a bunsen burner. To spread cells, just keep a bic lighter handy and a small beaker of ethanol (from CVS). Dip your inoculating loop in the ethanol, then just do a quick light to burn that ethanol off. Keeps it sterile. 

I use GoldenGate enzymes. No need for PCR reagents. I have 5 enzymes - T4 Ligase, T4 PNK, SapI, BbsI, and BsaI. About $250 off of NEB. I don't like PCR for several reasons. However, if it fits your fancy you can get those enzymes for a couple hundred more bucks.

I don't need microscopes. Why would I need a microscope? Why would I need a FEW microscopes? Here's a tip - learn what your bacteria smell like. I recall one time I caught contamination of my yeast stocks because I took a whiff of the overnight and it didn't have that familiar baker's-yeast smell. Bacillus subtilis is quite easy because it smells so much like dirty feet. E.coli also has a certain smell. You have a great biochemical sensor built into yourself! Use it!

List:
Enzymes : $250
Incubator + shaker: $150
Centrifuge : $100
PCR Machine: $150
Air filter: $150
Glassware: $100
Pipettes: $150
Disposables: $100
Media: $100
Other expenses (tables, boxes, tube holders, etc etc. Everything else you can think of): $250
---
Total: $1500

That seems expensive, but remember I have a very complete lab that I am quite proud of, and probably spent around the range of $2500 on over the range of the last 5 years (high-throughput is expensive ): ). If you're clever, I bet you can cut costs but many hundreds of dollars, and get a cloning setup to do your specific experiment for about $500. If you need any advice, hit me up. But no, you don't have to be rich. I'm certainly not rich, just dedicated :)

If you're not sure how to get all that stuff, Josiah has made it quite easy at the-odin http://www.the-odin.com/genetic-engineering-home-lab-kit/ 

Otherwise, back to eBay!

Koeng


On Friday, February 23, 2018 at 9:32:46 PM UTC-8, Michael Flynn wrote:
Hi.

Does anyone have a home cloning setup - how are you doing it? Are you rich? You must have an incubator, a -20c and -80C freezer, maybe a hood, digestion enzymes and PCR reagents, and a few microscopes? 

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[DIYbio] Re: Home cloning setup

Hi Flynn, 

I actually do have a cloning setup at home. My lab is actually specifically made for cloning (I like to figure out how to clone things well while working on the Free Genes project, then implement those things at home). And no, I'm not rich. I rent a single small bedroom, which is both my lab space and my home. 

Incubators are cheap from ebay. ~$100. I put an orbital shaker I got for $50 into it, so now I have both a shaking and stationary incubator. You also don't really need an incubator... bacteria grow just fine at room temperature, albeit for longer times. I often grow my sporulating Bacillus at room temperature so they don't take up room in my incubator. 

I don't actually have any freezer room except a small box for enzymes that I keep in our main freezer that I share with roommates. Unfortunately, that also means I don't really have space to store any food, so I usually keep dry food at home for eating. If I had room, I'd pick up a $200 chest freezer from Home Depot, that could store all my stuff for the foreseeable future. Oh, the glories of having space...

I don't have a -80C freezer. Those things are expensive man! And large. Right now I just use competent cells as I make them. That is getting very annoying, so I plan on paying for storage of my competent cells at a local biohacking lab. @Sebastian has also reported that the cells stay good for a few weeks at -20C, so it is quite possible if I had a chest freezer I could just make new batches every other week. 

For long term storage options, I've found that cells last for about a year at -20c in glycerol. They start doing funky stuff after that, so if you regenerate stocks every 6 months, it should be fine. Or just store plasmid stocks. Another good method is to spin down the cells just like you are about to do a miniprep, then freeze those stocks. If you need the plasmid, go ahead and do a miniprep of that frozen stock. It works well after a couple of months, and I haven't tried a yearlong experiment yet. 

You don't need a hood. Why would you need a hood? You don't even need a bunsen burner! Pick up an air filter from amazon for about $150, and that will be MORE than enough. In addition, when you make LB and the dust gets everywhere, the air filter will make sure your room doesn't get all dusty. It also keeps things nice and sterile, just like a bunsen burner. To spread cells, just keep a bic lighter handy and a small beaker of ethanol (from CVS). Dip your inoculating loop in the ethanol, then just do a quick light to burn that ethanol off. Keeps it sterile. 

I use GoldenGate enzymes. No need for PCR reagents. I have 5 enzymes - T4 Ligase, T4 PNK, SapI, BbsI, and BsaI. About $250 off of NEB. I don't like PCR for several reasons. However, if it fits your fancy you can get those enzymes for a couple hundred more bucks.

I don't need microscopes. Why would I need a microscope? Why would I need a FEW microscopes? Here's a tip - learn what your bacteria smell like. I recall one time I caught contamination of my yeast stocks because I took a whiff of the overnight and it didn't have that familiar baker's-yeast smell. Bacillus subtilis is quite easy because it smells so much like dirty feet. E.coli also has a certain smell. You have a great biochemical sensor built into yourself! Use it!

List:
Enzymes : $250
Incubator + shaker: $150
Centrifuge : $100
PCR Machine: $150
Air filter: $150
Glassware: $100
Pipettes: $150
Disposables: $100
Media: $100
Other expenses (tables, boxes, tube holders, etc etc. Everything else you can think of): $250
---
Total: $1500

That seems expensive, but remember I have a very complete lab that I am quite proud of, and probably spent around the range of $2500 on over the range of the last 5 years (high-throughput is expensive ): ). If you're clever, I bet you can cut costs but many hundreds of dollars, and get a cloning setup to do your specific experiment for about $500. If you need any advice, hit me up. But no, you don't have to be rich. I'm certainly not rich, just dedicated :)

If you're not sure how to get all that stuff, Josiah has made it quite easy at the-odin http://www.the-odin.com/genetic-engineering-home-lab-kit/ 

Otherwise, back to eBay!

Koeng


On Friday, February 23, 2018 at 9:32:46 PM UTC-8, Michael Flynn wrote:
Hi.

Does anyone have a home cloning setup - how are you doing it? Are you rich? You must have an incubator, a -20c and -80C freezer, maybe a hood, digestion enzymes and PCR reagents, and a few microscopes? 

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Re: [DIYbio] Home cloning setup

You can probably skip the -80C for a while, not really necessary unless you want to keep bacteria from mutating or you have some RNA you want to keep for a long time.

-SG

On Fri, Feb 23, 2018 at 9:32 PM Michael Flynn <mflynn210@gmail.com> wrote:
Hi.

Does anyone have a home cloning setup - how are you doing it? Are you rich? You must have an incubator, a -20c and -80C freezer, maybe a hood, digestion enzymes and PCR reagents, and a few microscopes? 

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