First ever project suggestions? - Starting genetic lab for hobby purposes. - (Jun/05/2015 )

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.  

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.

Artemis2007 on Thu Jun 11 20:07:15 2015 said:

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. 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.

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..

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

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.

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.

bob1 on Sat Jun 27 23:51:43 2015 said:

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.

Sincerely, N00b.

a -22°C freezer is ok.

It will be ok for shorter term storage of competent cells, but I guess you are not going to store huge amounts of them for a long time.

Forget the dry ice, thats too expensive for long term storage, you could use it for a short term, but -22°C will do as well.

pito on Sun Jun 28 17:11:09 2015 said:

a -22°C freezer is ok.

It will be ok for shorter term storage of competent cells, but I guess you are not going to store huge amounts of them for a long time.

 

Forget the dry ice, thats too expensive for long term storage, you could use it for a short term, but -22°C will do as well.

Phew! That really saved my day. However... please define "short term"? Is that like... 2 weeks, 2 months, 6 months? what? :)

hard to tell..

Most people keep competent cells in -80°C freezers, however some keep them in -22°C, but for how long? I do not know.

In most labs they just put them in the -80°C...

THe general idea or rule is to store them in -80°C freezers (or lower) and most people do not wonder about "what if". However: I know some keep them in -20°C and it worked.

Maybe the efficiency will be lower, but for your this is better than nothing at all because a -80°C seems to be out of the question and working with dry ice is also not really a possibility for long term storage.

Perhaps this can be a first experiment for you: making cells competent, transform them with a plasmid and store the rest of the cells in the -22°C and redo the experiment every week to see how good it works...

You can even try out different ways to make cells competent! This is even something that can be published! A lot of fuzz is still going around on competent cells and how to prepare them!

pito on Sun Jun 28 18:19:37 2015 said:

Perhaps this can be a first experiment for you: making cells competent, transform them with a plasmid and store the rest of the cells in the -22°C and redo the experiment every week to see how good it works...

You can even try out different ways to make cells competent! This is even something that can be published! A lot of fuzz is still going around on competent cells and how to prepare them!

Great idea! I will totally do that...

http://schneider.ncifcrf.gov/methods/TIBS/feb96.txt

you can start with this link, to find out what method you would prefer. I also attached two papers mentiond in that link. So you can , for example, pick one of those methods to prepare your competent cells.

It could be a nice experiment to perhaps try both methods (make cells competent with both methods), then transform with a plasmid and check for the efficiency.

And store competent the cells in -22°C  and redo the transformation every week and check how the efficiency is influenced by the storage time...

This is just a simple project (also pretty cheap) to start with.

Perhaps you can do it with more than 1 type of cells: eg. E. coli top10, E.coli dh5alpha, and...

it could already be a nice experiment and its pretty easy and cheap to try/do.

All you need are the cells and the plasmid!

(and the reagents of course)