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First ever project suggestions? - Starting genetic lab for hobby purposes.


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#1 Dr. N00b

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Posted 04 June 2015 - 11:53 PM

Hello everyone!

 

I've over the course of the last couple of years picked up an interest in genetics, and I am currently setting up a lab so that I can do some experiements as a hobby.

I am a bit unsure as to where to start.

 

Alot of my equipment is on the way from various countries as I write this, and within three to four weeks I will have a fully equipped lab. I have access to a PCR Machine, microsentrifuges with RPM's ranging from almost nothing to 15000, incubators, micropipettes, electrophoresis equipment, microscopes ranging from low magnification to 2000x oil immersion, bright-field and dark-field. I've even gotten my hands on a 2nd hand Bio-Rad Helios Gene gun...

 

So - I need something relatively simple as a first project. I thought "why not bacteria" - since the incubation time is quite short.

 

I've seen some images of people making paintings or drawins in petri dishes using e coli. Would this be a fairly straight forward task?

 

Or perhaps there is something else that would be visually interesting that I could do - that would require less skilled hands?

 

I would appreciate any input on this matter. :)

 

Sincerely, N00b.


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#2 pito

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Posted 05 June 2015 - 04:54 AM

Hello everyone!

 

I've over the course of the last couple of years picked up an interest in genetics, and I am currently setting up a lab so that I can do some experiements as a hobby.

I am a bit unsure as to where to start.

 

Alot of my equipment is on the way from various countries as I write this, and within three to four weeks I will have a fully equipped lab. I have access to a PCR Machine, microsentrifuges with RPM's ranging from almost nothing to 15000, incubators, micropipettes, electrophoresis equipment, microscopes ranging from low magnification to 2000x oil immersion, bright-field and dark-field. I've even gotten my hands on a 2nd hand Bio-Rad Helios Gene gun...

 

So - I need something relatively simple as a first project. I thought "why not bacteria" - since the incubation time is quite short.

 

I've seen some images of people making paintings or drawins in petri dishes using e coli. Would this be a fairly straight forward task?

 

Or perhaps there is something else that would be visually interesting that I could do - that would require less skilled hands?

 

I would appreciate any input on this matter. smile.png

 

Sincerely, N00b.

yeah, making those colored plates is fairly easy to do.

Al you need is some bacteria that are fluorescent... you can buy those or why not, make them yourself...

All you need is some save E.coli strain and some plasmids with the required genes.

 

See here for some information: http://14ilcatchicag...19_archive.html


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Dr. N00b

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Posted 05 June 2015 - 07:09 AM

 

...or why not, make them yourself...

All you need is some save E.coli strain and some plasmids with the required genes.

 

See here for some information: http://14ilcatchicago.blogspot.be/2014_07_19_archive.html

 

Very interesting!

 

I have been looking into this CHROmagar and briefly thought of ways to do it. From what I understand, with chromagar one makes a nuitricious agar using this substance, then grows the bacterial culture on it - and they will display the color chosen? If so - I guess I could make several agar plates, grow e-coli on them, then use the various e-coli colonies to create the final image?

 

About the E-coli strain and plasmids though.. I guess I need to make the plasmids myself? I mean, get a gene coding for fluorescense, create a plasmid vector, inject into E-coli and grow a culture? One for each color?

 

am I even slightly on the right track here? :)

 

Sincerely, N00b.


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#4 pito

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Posted 05 June 2015 - 08:41 AM

you do not need chromagar. Just regular medium will do.

You do need a blacklight to see the fluorescence!

 

And yes: all you need is a plasmid that expresses the GFP!

(and a regular E.coli to express the GFP/plasmid)

 

If you can find one with , eg, GFP you could use this plasmid for GFP and look around for other variations, like YFP and replace the GFP in your plasmid with YFP (or just use the YFP plasmid if thats possible)

 

And yes, you just grow each of them seperately and then you restreak them forming an image on a novel plate.

 

 

 

...or why not, make them yourself...

All you need is some save E.coli strain and some plasmids with the required genes.

 

See here for some information: http://14ilcatchicag...19_archive.html

 

Very interesting!

 

I have been looking into this CHROmagar and briefly thought of ways to do it. From what I understand, with chromagar one makes a nuitricious agar using this substance, then grows the bacterial culture on it - and they will display the color chosen? If so - I guess I could make several agar plates, grow e-coli on them, then use the various e-coli colonies to create the final image?

 

About the E-coli strain and plasmids though.. I guess I need to make the plasmids myself? I mean, get a gene coding for fluorescense, create a plasmid vector, inject into E-coli and grow a culture? One for each color?

 

am I even slightly on the right track here? smile.png

 

Sincerely, N00b.

 

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 Dr. N00b

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Posted 05 June 2015 - 10:41 AM

...And yes: all you need is a plasmid that expresses the GFP!

(and a regular E.coli to express the GFP/plasmid)

 

If you can find one with , eg, GFP you could use this plasmid for GFP and look around for other variations, like YFP and replace the GFP in your plasmid with YFP (or just use the YFP plasmid if thats possible)

 

And yes, you just grow each of them seperately and then you restreak them forming an image on a novel plate.


 

Ok...

 

So now all I need to figure out is how to make a plasmid, and where to find a GFP / YFP and then making the plasmid into a vector. smile.png I saw a video on e-coli and vectors a while back, but the details escape me. rolleyes.gif

 

I searched for GFP's and found a whole color pallet commercially available. They are complete vectors ready for use with a variety of bacteria. Among them ofc. e-coli.

However, these "kits" are expensive (225USD a piece).

 

I see Bio-Rad has a classroom kit for something similar. Perhaps it would be a good idea for me to go through one of these first. biggrin.png


Edited by Dr. N00b, 05 June 2015 - 10:41 AM.

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#6 pito

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Posted 05 June 2015 - 10:58 AM

Yes, it can be expensive...

What you could do is ask for some plasmids by sending emails to certain labs.. Some labs just send them for free (sometimes) other labs just ask you to pay for the mailingcosts, some labs just ask you to pay a small fee for the work when they have to send/prepare the plasmids...

You can get it pretty cheap in the end if you look a bit!

 

The kit I do not know it...

But could work yes.

 

In the end its not that hard: all you need is an e.coli strain, not hard to get.

Some plasmids (vectors witht the right GFPs on it): you should be able to find them somewhere too..

And you are ready to start!

Of course: if you really want to alter the plasmids, do some genetic work, you need to buy restriction enzymes and stuff.. that can get pretty expensive in the end!


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#7 Dr. N00b

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Posted 05 June 2015 - 12:19 PM


Of course: if you really want to alter the plasmids, do some genetic work, you need to buy restriction enzymes and stuff.. that can get pretty expensive in the end!

I kind of want to do most of the work myself. If not I can't really say "I've done this"..  laugh.png 

 

I found restriction enzymes though. The prices dont look half bad if you ask me. But then again, I dont know how many of them I will need for any one single operation.

 

Where does one ordinarily harvest e.coli? Just leave some raw chicken meat in room temperature, swab it, rub it in a petri dish, incubate and hope for the best?

 

It would be interesting to use several common bacteria for this so that it is not only e.coli. smile.png

What other easilly obtainable bacterias are there that I can harvest from say food, fecies, soil etc?


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#8 pito

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Posted 05 June 2015 - 12:48 PM

 


Of course: if you really want to alter the plasmids, do some genetic work, you need to buy restriction enzymes and stuff.. that can get pretty expensive in the end!

I kind of want to do most of the work myself. If not I can't really say "I've done this"..  laugh.png

 

I found restriction enzymes though. The prices dont look half bad if you ask me. But then again, I dont know how many of them I will need for any one single operation.

 

Where does one ordinarily harvest e.coli? Just leave some raw chicken meat in room temperature, swab it, rub it in a petri dish, incubate and hope for the best?

 

It would be interesting to use several common bacteria for this so that it is not only e.coli. smile.png

What other easilly obtainable bacterias are there that I can harvest from say food, fecies, soil etc?

 

 

NONO!

You do not just take e.coli from "raw chicken"!

You need a non pathogenic labstrain!

For example: K12 strain or any other labstrain that is safe to use.

 

You can find other bacteria as well, labstrains that are safe to work with.

 

Just start with safe labstrains rather than trying to find your own strains somewhere.. like food or fecies...

Thats a bit dangerous...

 

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#9 Dr. N00b

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Posted 05 June 2015 - 01:33 PM

NONO!

You do not just take e.coli from "raw chicken"!

You need a non pathogenic labstrain!

For example: K12 strain or any other labstrain that is safe to use.

 

You can find other bacteria as well, labstrains that are safe to work with.

 

Just start with safe labstrains rather than trying to find your own strains somewhere.. like food or fecies...

Thats a bit dangerous...

 

Allright... First mistake already avoided. happy.png

 

Ok, there we go... I found the K12 strain for sale.


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#10 pito

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Posted 05 June 2015 - 09:38 PM

yes, its easy to find it and you only need to buy it (or request it somewhere) and you are good for ever! You can simply grow it and store it yourself later.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#11 Phil Geis

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Posted 09 June 2015 - 12:42 PM

Doc - as with a y lab, I suggest your 1st project should be the IQ, OQ and PQ that establishes your equipment as functional and running standards that establish your application as generating accurate and reproducible results.  



#12 Artemis2007

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Posted 11 June 2015 - 12:07 PM

It might be very useful to spend some time in a working lab, learning the basic procedures in real life. Bench work doesn't always match the book and usually includes valuable information that no book has. Many research facilities have volunteer programs which could be a way for you to get started.



#13 Dr. N00b

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Posted 27 June 2015 - 10:00 AM

It might be very useful to spend some time in a working lab, learning the basic procedures in real life. Bench work doesn't always match the book and usually includes valuable information that no book has. Many research facilities have volunteer programs which could be a way for you to get started.

 

I understand that. However - I am not affiliated with any university or school, and I do not work in a profession that in any ways entails me rubbing shoulders with lab-people, if that is an acceptable term. smile.png I work in Aerospace with high precision tools. Far far away from any genetics lab.

 

I live in Norway - and in order to even set foot in a lab here you need to either be an enrolled student at an apropriate university, or a qualified lab-assistant or the likes, hired to actually work in a lab environment. If I were to e-mail a lab or two at a hospital or university or what not - I'd most likely be met with silence, or a polite reply that "sorry, but we are unable to offer you any such assistance".

 

The other day I made a few calls to the local hospital(s) that had suitable labs. I asked about lab strain e.coli and whether or not they had any. I was put through from phone to phone until I finally met someone who could not help me. They had lab strain e.coli on ice though.. They just couldnt give or sell any to me.

 

So... to the point: I am completely on my own here, and I will continue to be on my own.

 

I do however - thanks to a very friendly and helpful customer relations worker at Bio-Rad Life Systems - have a customer account with them.

Which brings me to the reason why I looked through my posts and started writing here. smile.png

 

If I am going to get any e.coli, I need to buy it from an actual provider. Bio-Rad is such a provider.

Now, this stuff is expensive... And I am thinking, which e.coli could I buy from them and "keep alive" in a large, or several colonies by myself to have a relatively endless supply of them?

 

So far I have been recommended these two products:

 

Subcloning Efficiency™ DH5α™ Competent Cells

and

One Shot® TOP10 Chemically Competent E. coli

 

the first one is obviously the most affordable to me, but at my current level I am unable to distinguish the different versions from eachother to find the most suitable allrounder.

What I want to do is created plasmid vectors, insert separated gene into vector, infuse e.coli with this vector and have them express - say a green fluoresence protein. Which product do I go for?

I know I can buy ready-to-go plasmid vectors, but I want to learn how to do it myself... so.. smile.png

 

Oh, and welcome to the world's smallest lab. (see attachments).

Lab (1 of 3).jpg

Lab (2 of 3).jpg

Lab (3 of 3).jpg

It is starting to look like something. I still need to get an autoclave amongst other things. I will continue to build on this during the next few months. I do not own a house, so this is all the space I've got to work with. I got inspired by the bio-hacking community and went all out. :)

 

Sincerely, Dr. N00b.


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#14 bob1

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Posted 27 June 2015 - 03:51 PM

That looks fairly well equipped. For the bugs I would try sources such as the ATCC (atcc.org) and the European equivalent (seeing as you are in Norway), the ECACC. These are (at least the ATCC is) not-for-profit organisations and may be able to send you some stuff for free. Either of the recommended strains will work. DH5a is the most commonly used strain, and works fine for just about every application. These stocks are supplied as already competent stocks, so you don't have to make them competent before transforming with the desired DNA. These would need to be stored at -70 C or thereabouts (dry ice would work too). If you can get some ordinary DH5a or Top10, you can easily make them competent - check the protocols on openwetware.org. With regards to the plasmids, you could try Addgene.org, they are similar, but more of a repository for linking researchers with each-other, so much of the stuff on there would need a memorandum of understanding to get it sent to you. Otherwise there may be someone on here who would be willing to send you some plasmid dried onto filter paper, but you just have to work out which plasmid you want, and if someone has some available.

#15 Dr. N00b

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Posted 28 June 2015 - 09:05 AM

That looks fairly well equipped. For the bugs I would try sources such as the ATCC (atcc.org) and the European equivalent (seeing as you are in Norway),

 

...These would need to be stored at -70 C or thereabouts (dry ice would work too). If you can get some ordinary DH5a or Top10, you can easily make them competent

 

...Otherwise there may be someone on here who would be willing to send you some plasmid dried onto filter paper, but you just have to work out which plasmid you want, and if someone has some available.

 

Thank you! My lab is my pride and love these days. I cant wait to get some more equipment in there though.. So damn expensive. Need to put in more extra hours at work... :P

 

As for -70 C storage. I only have a -22 C freezer, and those super-cold lab freezers are far out of my budget range. :( However, you mentioned dry ice... Now, could I store that in a regular heavy duty ice-box? I am unable to find dry-ice specific ice boxes, except for a couple of really big ones on e-bay that will not ship to Norway.

If I keep the lid unfastened, the pressure built up inside should be able to vent every once in a while, shouldnt it? I am currently looking at something like this.

Its not made for dry ice, but perhaps I could get a bigger one, and put some more polystyrene in it to insulate even more... And just not lock down the lid, allowing it to vent whenever the pressure is high enough to push open the lid ever so slightly?

 

As for getting help from forumites to secure some e.coli - I have allready been contacted by a friendly soul. ;) We'll see how this goes. :)

 

I'd also like to add that I really appreciate all the feedback I've gotten and continue to get on my posts on this forum. People eager to help a stranger in need is a rare thing in todays society. I suspect I shall gain a great deal of knowledge from this forum. happy.png

 

Sincerely, N00b.


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