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pito

Member Since 27 Jan 2009
Offline Last Active Jun 17 2013 09:20 AM

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#153733 New to the field - literature advice needed

Posted pito on 14 April 2013 - 10:23 PM

aerkenemesis, on 14 April 2013 - 01:24 PM, said:

pito, on 14 April 2013 - 10:25 AM, said:

Just wondering: why are you doing this?

I can't see why I would need a reason to take part of the seemingly classified literature that goes with this subject.

The literature I can understand, but I wonder what you really want to do?
Are you really going to try to inoculate your garden with it?

And do you have any basic microbiology books? Because I can give you some titles that are avaible online.
But I wonder how much you allready know.

#152659 Same first name and second name of two authors in one paper???

Posted pito on 21 March 2013 - 09:37 AM

mansi368, on 21 March 2013 - 09:28 AM, said:

Hi..

This is heights of coincidence but there are two people of same first name and second name in my lab. Both of them worked equally for one publication and morally both own it..

The problem is how to write their names as both first name and second name are same??? The computer everytime takes it as an anomaly and erases the name written twice......

What to do please suggest

Thanks

Use an initial of their middle name? Even if one of them has that.. you can use it.

Or dont put them next to eachother?
Or use a sign next to their name that means: contributed equally for example *1 next to the first name and *2 next to the second

#148638 Recreate original plasmid by cutting out insert

Posted pito on 23 January 2013 - 06:51 AM

schaapje_86, on 23 January 2013 - 06:03 AM, said:

Maybe this is a stupid question, but I am going to ask it anyway:

I would like to cut out an insert from my plasmid (pET28c) between restriction sites Nhe1 and Not1 and ligate the plasmid back to it's orginial state without the insert (no I don't have the original plasmid anymore). However, obviously these restriction sites don't generate compatible sticky ends. Does anyone know how to recreate this plasmid?

Many thanks!

A hint:
Klenow polymerase
Making blunt ends.
Do a google search, you should be able to find helpfull information.
If something is not clear, just ask, but I think if you look those two things up, you will know what to do!

#147661 Restriction Enzymes

Posted pito on 08 January 2013 - 02:00 AM

Pangea, on 08 January 2013 - 01:56 AM, said:

Ok , lets say i want gene sequence of an RE from IMAGE consortium. And produce my own RE in E.coli. I am no considering the cost and sso on.. . Question is if its practcally possible in the LAB.

sure.

Its possible but not practical Posted Image

You simply need the DNA sequence of the restriction enzym with the promoter etc... there is no reason that it would not work, but if you have to collect the enzyme/purify it etc.. it will take a lot of time, money ...

BTW: check this topic http://www.protocol-...ing-polymerase/
its not about a RE , but the same goes for you. Altough, your would be harder to collect/purify

#147516 Ethidiumbromide

Posted pito on 05 January 2013 - 07:22 AM

SYBR Green for example..

You should be able to find them yourself....
However keep in mind that some of the alternatives seem to be more "dangerous" then ethidium bromide itself.

PS. the horror stories about ethidiumbromide are really over the top and not true at all. Many people seem not to be able to search for information themself and just accept "stories" others told them.

#146363 Animal-Derived Laboratory Reagents

Posted pito on 06 December 2012 - 12:53 PM

southtowns18, on 06 December 2012 - 10:22 AM, said:

I have a question about animal-derived laboratory reagents and products. I’m working in a strictly in-vitro lab that focuses mainly on western blots and flow cytometry. I was wondering the cost to animal life associated with these reagents. These include:

FBS (fetal bovine serum) & BSA (bovine serum albumen): I figured these products are likely a byproduct of the slaughterhouse industry and therefore the animals are being slaughtered more for the meat than to get the blood to make these products. Am I right in thinking this?

Primary & secondary antibodies (raised in mouse, rabbit, goat, etc.): From what I understand there are three main types with differing degrees of cost to animal life

In vitro Monoclonal antibodies: Once the hybridoma is created they can be grown indefinitely in vitro so there is minimal cost to animal life. Am I right in thinking this?
In vivo (ascites) monoclonal antibodies: Does an animal have to die every time I place a new order for an antibody?
Polyclonal antibodies: Again, does an animal have to die every time I place a new order for an antibody?

Sheep’s blood agar: Unlike the bovine products, I wouldn’t think sheep are being killed for the meat so maybe they are being primarily slaughtered to generate these lab products. Am I right for thinking this?

I’m asking these questions because I enjoy working in the lab but just want to know the cost associated with the products I use in regards to animal life. Also, can you think of any other animal-derived reagants/products that I left out that could be used in in-vitro experiments?

Thanks for any help you can provide.

Why do you think sheeps are not killed for the meat?
Here its pretty normal to eat sheep.
But most sheeps are not killed in general because the wol is worth a lot more.
They just take some of the blood... but they do not need to kill the sheep for it.

Anyway, if you really want to know about this: contact the people where you buy the stuff.
Some companies have strict rules about this. Some will, for example, not work with animals being killed for just 1 product.

And in general: does an animal need to die? No!
For example horses are used to produce antidotes against snakevenom.. they dont kill the horse for it.

Also, http://en.wikipedia....al_bovine_serum, for you question about FBS en BSA

#144762 Co-worker hides mistakes, reports questionable data

Posted pito on 07 November 2012 - 04:07 AM

One advise:

make sure you have anough of "proof".
If you cant catch her in the act, make sure you have "proof" that shows something is going wrong.
Use this to in a discussion with your boss.

And one hint: you dont need to accuse her right away, just collect the "proof", ask your boss to have a chat with you to discuss some strange things (without pointing the finger at her).
You can genlty mention her name , for example: we noticed some strange things with samples A and B and with the testsresults from test Y , perhaps we can debate what went wrong? Normally your boss will then ask who did those tests ....
See what I mean..
Dont start accusing her right away yourself because it can backfire + if you cant really catch her..

Also: if she comes in late and leaves early.. its something the boss needs to check, not you.
I know its not honest, but if you start complaining about it...

#144436 Journal of Biochemistry and Molecular Biology Education (BAMBED)

Posted pito on 01 November 2012 - 03:44 AM

Trof, on 01 November 2012 - 02:51 AM, said:

Thanks, at moment I have enough time I will start to stick the useful info to the top.
(unfortunatelly for me, I don't have access to this journal)

I have acces to it. Is there a specific paper you are looking for?

BTW: I find it pretty idiotic that a journal like this is not open acces!

Its not about publishing researchresults, so I do not understand it why this paper is not open acces nor do I understand that it has an impactfactor to be honest.

#140288 10 millimolar (mM) to 250 micromolar

Posted pito on 30 August 2012 - 06:08 AM

ascacioc, on 30 August 2012 - 01:51 AM, said:

Maybe you do another thread that we can pin on top of the general techniques forum for having it as a reference forever and ever and not have the same explanation repeated over and over (I am tired of typing) and then we can just refer to that thread, don't you think?

Andreea

You are the moderator, I am not.
Do what you think is a good idea.
Perhaps its a good idea to make 1 general post with some simple examples in it and pin this somewhere so they can always check this post.

I can only tell you that I have seen hundreds of these kind of posts. People suffer a lot with elementary dilution problems. And almost everytime its the same problem: they have heard of a formula or they even know CV = CV , but have no idea how to use it or what it means.

#139881 Deletion Mutant - Evolution

Posted pito on 23 August 2012 - 11:29 PM

ascacioc, on 23 August 2012 - 01:43 PM, said:

wow...quite a lot happened here while I was in the lab. Just a few words to the first post after mine:
somatic hypermutation is epPCR: in the lymphoid system the mutation rate is 10^6 higher than in the normal cells; epPCR is amplification with a high mutation rate. And this is not semantics.
I give you that at least: indeed VDJ recombination is a totally different system than DNA shuffling....even though parallels can be drawn.
Both DNA shuffling and epPCR are very controlled: now, if you use genome shuffling a la Stamer (first protocol ever published) you indeed cannot control it, but recent protocols for both epPCR and DNA shuffling are very well characterized and you can predict what you have in the end in the test tubes. There are programs and algorithms/scripts that do that for you. I worked developing some myself during my master thesis...and there were unexpectidily quite good in telling me what some other people get in their test tubes which means that we did not have so many uncontrolable stuff happening in the test tube (as I thought in the beginning of my masters when I was like you: riiiight you can control it)
I did not link DNA shuffling and epPCR more than the basic two protocols used in directed evolution. They are totally differently.
Of course that what we call directed evolution in the tube would not happen in nature: depends on where the selective pressure lays. I mean: if I evolve for example glucose oxidase to be more active to use it for a biofuel (real project on which people are actually working) you will not get the same things as in nature because while you are lowering the Km and making the kcat higher by directed evolution for you purpose, maybe in real life it is not good for this enzyme to use up in a fraction of second all your glucose in an organism and release H2O2 like tons of it in the same fraction of second because the organism will starve and will be killed by the toxicity immediatelly. However, it matter where you put the selection pressure: if you choose a smart selection pressure to keep the good mutations that are beneficial for an organism, you can simulate evolution.

And about not agreeing with that sentence: well, you are not agreeing with an entire field.

@prabhubct: fast-backwarding: I do not know how the selection pressure would work to fast backward something. I only know how to improve stuff, not how to make them worse Posted Image

Andreea

you are right about, my mistake.

Quote

somatic hypermutation is epPCR: in the lymphoid system the mutation rate is 10^6 higher than in the normal cells; epPCR is amplification with a high mutation rate. And this is not semantics

I misread your first post and I was talking more about what went on before the mutation.

Its indeed as you said: after binding antigens this starts, but the receptors themsellf (the first ones) are allready made at that time. I was thinking you ment the entire proces from the start which is of course not right.

PS.: you state that I am not agreeing with an entire field, I dont know if you are an immunologist, but if you are you, should know that even they arent agreeing completely, a lot of questionmarks are still out ther in that specific field. Its a pretty new field. Altough they do seem to agree on the proces, but how it happens exactly etc... still a debate going on.

about the followig:

Quote

Both DNA shuffling and epPCR are very controlled: now, if you use genome shuffling a la Stamer (first protocol ever published) you indeed cannot control it, but recent protocols for both epPCR and DNA shuffling are very well characterized and you can predict what you have in the end in the test tubes. There are programs and algorithms/scripts that do that for you. I worked developing some myself during my master thesis...and there were unexpectidily quite good in telling me what some other people get in their test tubes which means that we did not have so many uncontrolable stuff happening in the test tube (as I thought in the beginning of my masters when I was like you: riiiight you can control it)

This is just it: either you control it and then you are not really working with evolution, because evolution is not really controlled at all.. (at least not at the level people control it in test tubes during this kind of experiments). Or you cant control it and you do a completely random shuffling and then there is no link with evolution at all because you shuffle it way more then nature.

The thing is: you can control it because you set certain values yourself at the start! You start controling it yourself ..... thats not what you call evolution.
You pick the strains, you pick enzymes (sometimes, or more often then not, they work with restriction enzymes not even natural to the organisms), you pick the working temp, the media, ... you control a lot of the parameters...
BTW: the entire discussion here is nothing more then what physicist (and biologists) are debating for yours: chaostheory, randomness etc
Or on a footnot: what religious people are also stating.

Also, I like what you said about "evolving", but this sentence kinda breaks it down:

Quote

However, it matter where you put the selection pressure: if you choose a smart selection pressure to keep the good mutations that are beneficial for an organism, you can simulate evolution.

All you can do is mimick what you think happens in nature, you define those boundaries.. A lot of "you" here and not a lot of "nature".

A lot of the work done on micro-organisms in the field of "evolution" is not really following the debate about what evolution now really is. The definition of evolution is changing almost every 10 years and even when people (like you?) speak about fast foward evolution in bacteria/yeast, we arent even able to correctly link protists based on evolutionary mutations etc...
All we do in the lab is cause mutations to occur , have genes rearranged , check for "better" organisms we can use or "new" organims and then we call this evolution. There are papers out there describing the "evolution" of a bacterium towards a better bacterium to bio-degrade some toxicA, is this really evolution? Is putting genes from yeast 1 in bacterium 4 and then caling this evolution really correct?
Dont forget that in many shuffeling experiments you mix genes from different yeasts ....
Or you push the organisms towards a certain evolution.. like you allready said: you select based on what you (we) want... weird view of evolution to be honest.
Your sentence said it, it makes my point:

Quote

@prabhubct: fast-backwarding: I do not know how the selection pressure would work to fast backward something. I only know how to improve stuff, not how to make them worse Posted Image

You only know how to improve stuff.. not to make them worse.. well there you go... evolution doesnt work like that at all.
what we call improving is based on our needs, and the definition of making things worse is also based on our standards. Dont forget that in the history of evolution a lot of the so called "bad" evolutions turned out to be good.

Evolution seems to be a very wide definition for many biotechnologists.

I think there should be some new definition or general agreement on what we do: evolve things like we want vs (real) evolution.

#139825 Deletion Mutant - Evolution

Posted pito on 23 August 2012 - 04:05 AM

prabhubct, on 22 August 2012 - 09:58 PM, said:

pito, on 22 August 2012 - 12:04 PM, said:

ascacioc, on 22 August 2012 - 11:06 AM, said:

@Pito: genome shuffling happens in nature: V(D)J recombination is DNA shuffling; this is the way your body makes antibodies by combining the alleles in the MHC class I loci; moreover in the next step, your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus.

The point of epPCR is that it actually recreates the same most probable mutations/errors done by DNA polymerases in bacteria. There are tons of papers calculating the probabilities of certain transitions/transversions and the hot spots for mutations and they are pretty much the same no matter what polymerase you are using. So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube. (some people use this cliche over and over again in the directed evolution field)

@prabhubct: if you would like to read more about directed evolution: http://www.sesam-bio...ected-evolution A pretty good review of the state of the art 2-3 years ago (I wrote it:P) It also has references to material that paraallels directed evolution to natural evolution.

Andreea

Of course genomeshuffeling happens in nature, but there is a difference between what you call genome shuffeling that happens in nature (there is a system behind it, I am talking about the VDJ recombination now) and genome shuffeling we do in the lab.

And I do not agree with what you state here, its a bit "risky" to state it like this: "your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus"
This is not entirely correct.
The body does not "evolve" like you state it.
The body has allready a bunch of antibodies present, by pure luck a few of those happen to bind the antigen, because they do, they will be favored and other that do not bind will not (or less) by enriched (recreated) by the body. This is called affinity maturation of antibodies. This happens because the B cells with the best binding receptors will bind the anitgen and will be selected (and survive/multiply and pass on their genes) because they are able to bind the follicular dendritic cells (those will bind the antigen, so the B cells bind indireclty) this is how affinity maturation happens and how "evolution" of antigens work. ANd yes, due to simple mutations in those cells, you will also create better cells in the end. But its bit different from just stating what you stated.

But at the start: its all a random proces, your body just "creates" random antibodies by VDJ recombination (random; but with a certain system).

This is completely different from the genome shuffling you are speaking of. In genome shuffling you cut DNA and create random new pieces of DNA by extending them again, ligating them.
+ genome shuffeling and then compare it with VDJ.. a bit weird esp since I was talking about bacteria, but perhaps I did not state this clear enough.
In bacteria there is no such thing as VDJ recombination.
And I dont like to link systems we use in bacteria/yeast for human or animal systems/genes.

Also: epPCR can indeed be used as a tool to study what happens with bacteria/yeast for example and you can indeed call it fast evolution, but it doesnt really represent 100% what happens in nature.
Its just a tool to cause mutation, nothing more.
ANd yes, you could state that those mutation (or some) would indeed also happen in nature , but nature is far more complex.

Also: linking genome shuffeling and epPCR is a bridge to far for me.
epPCR is much more controlled while genome shuffeling is (or can be) less controlled and you can get stranger results.
Altough, in the end its all about how you define certain stuff.

Also, and you said it yourself: its called "directed" evolution, thats just it: we (the researchers) direct evolution.. you cant simple say: aha, this is what would happen in nature.
Its a bit easy to state that.

What you create in the lab, I wouldnt call it evolution. I would call it: an observation of changes in DNA that cause a certain (observed/measured) effect, which in the end could indeed be a representation of a certain (possible) evolution.
You need to keep in mind that many of the so called "evolutions" caused by direct evolution techniques would not survive in nature or stay evolved like this because we want this evolution and keep in, while in nature the evolution might we "stupid" and not wanted and thus be lost in the end.

But this is more about semantics.

But do not agree with "directed evolution is defined as fast-forwarding natural evolution in a test tube" because for me this is not correct. A lot of the so called "direct evolution" are no evolutions that would happen in nature.

thanks.
@ pito : as you said ''epPCR can indeed be used as a tool to study what happens with bacteria/yeast for example and you can indeed call it fast evolution, but it doesnt really represent 100% what happens in nature..'' i agree with you as directed evolution could not represent 100% of nature. But it do represent some probabilistic value for evolution if we do want to believe in transition, transversion, recombination as means of evolution.

Yes,it does represents some possibilities.
Thats the idea behind it, but you should never forget what you are really doing compared with evolution

#138908 How delete survival gene in bacteria still keeping cell alive.

Posted pito on 03 August 2012 - 01:01 PM

prabhubct, on 03 August 2012 - 07:28 AM, said:

How can we select bacterial mutant on growth medium if survival gene is deleted. merely no colony to judge as gene is essential for growth may not be effective. Reason for no colony might be errors in experiment or homologous recombination problem.

what if survival product is not food but some internal machinery of bacteria say DNA polymerase which could not be supplied from outside.

You are right.

The problem with deleting or mutating an essential gene does indeed imply that the bacterium wont grow...
See the attachement on how researchers look for essential genes.
And check this: http://www.nature.co...p00125.html#/f2, check figure 1!

Also: to make sure its not because of the medium, you use the same medium as when the gene was not distrupted.

And about the internal machinery of bacteria not be supplied from outside: not sure what you mean by this... If it can not be supplied from outside when you altered the gene.. it will also not be supplied from outside without altering the gene (unless its a gene for a transporter that brings in , for example, a certain nutrient... but then again: it means that the gene you changed is essential for survival).

Attached Files

Song & Ko.pdf 255.47K 115 downloads

#137092 Phd topic

Posted pito on 06 July 2012 - 12:17 PM

What I do not really understand is the fact that you (the "student") are the one that is "finding" a good PhD topic.

Is this normal in your country?

In belgium its a little bit the other way around: professors find intersting topics and search for PhD people...
Its rare that students come up with their own ideas.. basically because students often arent realistic enough or dont know a lot or not enough to come up with ideas.

Anyway, it seems that you are going to do something with tropical animal husbandry? Why not check research facilities (in other countries) in this area and see what kind of topics they offer?
In belgium for example we have some open topics (still looking for students) , maybe you can get some inspiration from those?
If you want, I can give you some links and perhaps that could give you some inspiration or who know, perhaps you would even like one of them and come the belgium haha

#133454 Extracting DNA from fruits and Vegetables

Posted pito on 24 April 2012 - 11:35 PM

hsbiopeep, on 24 April 2012 - 04:14 PM, said:

I was given this topic by my biology teacher but I am not sure how I will be able to observe the differences between fruits and vegetables.

Will certain bands only be present in fruits rather than vegetables?

What I am trying to ask is how should I try to observe the differences between fruits and vegetables?

Its all you can do: use the RE and then watch what it gives on the gel.
I have never done this before, so not sure what to expect.
But indeed: the more the DNA is different, the more difference you will see in the bands...

Just test a few different kinds of fruits and test some vegetables, put them on gel (more then 1 sample per fruit/vege) and check the differences.
Check fruits vs fruits and fruits vs vegetables and vegetables vs vegetables

Also: be sure to test the tomato.. (look up: Nix v. Hedden) and see for yourself whether the court was right or not...
(be sure to try to find a definition on what a fruit or vegetable is..)
(==> also starting from this "definition" you should be able to find out whether there would be a big difference or not and what might be the cause of this difference on DNA level..)

#133302 Extracting DNA from fruits and Vegetables

Posted pito on 22 April 2012 - 11:26 PM

hsbiopeep, on 22 April 2012 - 04:04 PM, said:

pito, on 15 April 2012 - 10:32 AM, said:

Sure why not.
Hae III wiill cut a lot, so its possible you will get very small pieces of DNA.

So is it good to have small pieces of DNA or not?

Well.. it depends...
It all depends on what you want.

What I wanted to say with it is that you will get many small pieces.. so you will have a gel with many pieces.. rather then a few.. It could be more difficult to see differences ...
Also: small pieces means: watch out with the time you let the gel run.. the pieces could migrate off the gel.

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