Basic "Hello World" lab experiment - Help the newbie! (Jun/06/2010 )

Hello all,

The point of this topic is to request some guidance related to making the most basic procedures where one can manipulate DNA on every level without having any previous experience or know-how.

For example (I'm sure there are many other samples):
Take some bacteria from a rotting fruit or something like that, extract its DNA, somehow change the sequence and insert some code into it (for example a glowing gene) and then get it back in.

The guide will explain the basic tools and material required as well as the actual procedure.

If you have the know-how, even a basic top view (without much explanations, tough the more you share - the better) is a start - provide some model of the whole procedure and I will try to look around for the details and post it here - you tell me if I got it correctly.

Hi Yocttar -- welcome to the Bioforums!

Basically, you're asking how to start with an environmental source, and end up with a recombinant bacterium? Yikes -- what a huge question! It would take a whole textbook to answer such a question. Why do you ask it?

Basically, I learned biology, biotechnology and some basic biology course, I know the definitions but I don't know how to practically implement them, I study from home, never been in a bio lab.
My expertise are programming (just about anything, expert in C) and electronics (Altera, 8051, VHDL, orcad etc..), I know some basic mechanics as well (all those can be experimented and practised with software from home), my weak spots are chemistry and biology and I seek practical guidance!

I wouldn't mind to build or buy anything as long as it won't cost too much.

Yocttar on Jun 6 2010, 07:14 AM said:

I'm sorry but you'd need at least 3000$ to get the "basical" stuff for those kinds of experiments. And there's no way to do em without that stuff. Can you afford that?

alex84xela on Jun 7 2010, 03:15 PM said:

If it is essential and will get me where I want, then the answer is yes.

BTW, I try to understand the shortest way to achieve that goal...
Example: Generate a DNA strand using synthetic methods (which I don't know how yet) then insert it to a bacteria or algae (which I don't know how yet) then let it grow in some dish (which I don't know how yet) and then see the light in the end of the tunnel (which every one knows how!).

Few notes:

To get "some" DNA (what is it, an existing gene? non-existing (so how you know the sequence then?) a regulation sequence? your coded email?) there are various methods of obtaining it (depends on WHAT it is) and using de novo synthesis isn't a common or cheapest way to get it.
Usualy you want to put a sequence from one organism to another, so you use PCR (if it's up to few kbp) or restriction (if it's larger) to get a piece which you then ligate to a suitable vector (a plasmid usually, in the case of bacteria) (and again, what do you want your sequence to do, express? recombinate to bacterial chromosome?) and than transfect the plasmid to a E.coli strain first to multiply it, then isolate the plasmid and put it to your bacteria (non trivial step if your bacteria doesn't want plasmids) of just transfect the DNA fragment into the cell (even eukaryote, not sure about algae though) by other methods (virus, lipid vectors, electroporation,..?). You then may want your fragment to recombinate to host DNA. And then you should screen for succesfull recombinants (for this you need to add some detectable genes to your construct).

A few years work in a fully equiped lab under skilled supervision.

This is far from being a "basic procedure", that's advanced gene manipulation. Even the wide used and basic technique like PCR requires costly machine, pipettes and chemicals. It would be even cheaper, I think, to "lease" a time in some lab for that.

So I would ask, what do wish to achieve? What is it for? Why do want to do this?

For getting a basic grip of biotechnology at home, I would go for "DNA extraction at home" or something like that.

For a gene manipulation, there is no easy way like "Hello world" program.
-------------------------------------------------------------------------------------------------------------
Sub BioProjectOnLoad.GeneCloning

If funding < USD10000 Then
call ForgetAboutIt
ElseIf experience = 0 Then
call ForgetAboutIt
Else
Goto Plan_Project
End If

Plan_Project
If equipments = yes Then
call StartProject
ElseIf equipmentborrow = yes Then
call StartProject
Else
call ForgetAboutIt
End If

StartProject
If result = yes then MsgBox("Congrats")
ElseIf result =no then
call Plan_Project // this can be infinity if you choose not to give up
Else //give up finally
call ForgetAboutIt

ForgetAboutIt
Execute: changePlanAndBeHappy.exe
End Sub

Well though I don't know what exactly you want to do, and though I'm not sure if it's a good idea if someone wants to start a biotech lab at home in his or her kitchen to produce genetically modified micro-organisms (that then are perhaps later go in the sink), anyway I won't find it that expensive and difficult, if one starts with standard procedures and organisms such as E. coli.

There are starter kits for schools available, that contain all the necessary equipment (except some beakers, bunsen burners etc) and chemicals. have e.g. a look here, that is a bit more expensive and sophisticated...but even more simple and cheap kits are available, though only some standard experiments are possible.
If I remember right, some of the chemicals, many micro-organisms stock cultures, plamids etc are only sold to "trustworthy" organisations and companies, but not to private persons; that might be a major barrier...For other chemicals it depends on the country, some sell it without restrictions e.g. in pharmacies (as in my country), in others not...but DNA extraction is possible with household chemicals, as also Trof's google search shows...Some old and/or used lab equipment can be obtained at a more or less low price, ebay is a good source for that, even micro-pipettes and thermocyclers you can get there..

Thank you all for your answer.
The way I see it now is that I was aiming too high, the take home message for me (for now) is
"A few years work in a fully equipped lab under skilled supervision."

Anyhow, I just wanted a RepRap kind of thing for biology
Guess it's going to take a few years, But I will never give up!

BTW, I do understand all the ethical and biological hazards related to such work, I guess it's good that this is hard to achieve (for now).