ascacioc

Yes, probably the tag is hidden by the protein. You can try a C-terminal tag or a longer N-terminal His-tag. How long is your tag now?
Maybe you can try also to lower your imidazole concentration (~20 mM) during the wash step.

@ascacioc: As far as I know, the 100 ng of human gDNA in reaction is like golden standard. When I'm working with other templates I recalculate to equal number of target gene copies, because It's actually number of copies that matter (however I understand it's more complicated than this, since generaly more complex templates amplify differently). Usually using my web aplication for copy number.

But practically gDNA is not that sensitive to the exact amount in reaction. I usually don't even measure DNA before PCR and just put there 1 ul, it works It will work with 50 ng, 300 ng, 500 ng.. generaly not more than 1 ug. Since all isolation procedures and blood volumes we use are similar, the concentration doesn't vary that much, usualy it's around those 300 ng/ul. In that case I don't bother to dilute it for PCR and specific concenetration, because it doesn't matter. If there is extremely high concentration then I dilute, because reaction would fail.

Of course there are methods where the concentration should be adjusted, for any real-time quantitation generaly less than 100 ng is used, because it's very sensitive. Especially with SYBR assays the increased overal DNA concentration causes high background level fluorescence. For special aplications like HRM even less is used, between 5-30ng in reaction, all samples adjusted to the same concentration.

When you ask someone how much gDNA to put in reaction, he will usually tell you 1 ul as a joke. It's not a joke though

Well if God made Adam and Eve from clay I would try to found out where he get the nucleotides first, not talking about any rRNA...

In my oppinion it's not possible to take the Bible literaly. From that comes the simple implication, that people who take Bible literaly are wrong. Be it for any reason, that it was writen by God (he has strange sense of humor then, since there are contradictions and mistakes) was written by someone by direct inspiration from God (well he should have taken someone more copetent then).. anyway it means that Bible is not a perfect ideal absolutelly correct holly-spirited amazing wonder, but just a book.
I take it that christians finds the book quite interesting, inspiring and wise, the parables or so, why not, that is only the way to percieve it, I found wery amazing insipiring and wise the american remake of The Ring (I mean it, I made me thing really deeply about human nature and I came with a theory obout fluctuation of evil.. but about that maybe some other day..), thing it these thing we see between the lines are in part individuall, so for someone it's Bible, for someone Qur'an for someone esoteric literature and for someone it's the book  About the mole who wanted to know who pooped on his head.
So if someone tries to persuade me that only Bible is right or is the only source of morality and wisdom, I just woudn't agree. The christianity and religion in general doesn't have a monopoly for moral. Besides, I personaly tried to read the real Bible (I read the childrens version when I was young, with lot's of images and I thougt that Bible was sort of book with ancient stories) and I was repulsed, really, what they call a Gods superiority I just see arogance. In the Second part I just see the arogance multiplied, I've seen those missionaries telling people on the streets to read Bible. I did, and God... Jesus is a huge jerk! So what does it tell? Wrong translations from hebrew (or what language) they say.. well, lot's of people swear on their translations od Bible as it was a law.
So what does all this say? Nothing, that just I have to find interesting and moving things for me somewhere else than in the Bible, that's all. Bible is just not as unique. It's been massively advertized through centuries.

So basically I have no problem with any religious (or atheist for that matter) person who is not close minded or dogmatic or fanatic of any kind. Apart from that it is a personal thing. Most people who I know and have no problem with don't have those absurd christian (or other religion) oppinions about public matters, and don't take the holly textbooks literally, just as an inspiration (hopefull not of being a jerk). One friend told me about the christian unitarians groups he comes to meet (funny on the fact is, that he's actually jewish), and that he likes it there, because their motto is always doubt, always ask questions. I think that's not a bad move, if they take it seriously. People of this kind can be actually scientists without any problem, because they don't close they eyes from facts. I had one colleague and he once said, partly in joke, that there must be a God, because enzymes are such a wonder. Also the best-known astronomist of my country (you can say such local Carl Sagan) and member of a important skeptic group actually in private believes in God, and was repeatadly explaining that these doesn't exclude. None of those have any problem with evolution or the Big Bang, naturally.

Another question is a personal believe, where I came to conclusion that for me is any religion a merely unnecessary extension above things that are really important. It's like you need a driver to a very required part of hardware, and you can choose either to compile your own (linux style), or choose from existing driver by various companies, some open-source, some freeware, some paid.. and that religion or specifically christianity is like a 250MB HP driver, that installs many things you don't actually need in addition. So I go for the own-compiled driver, it may take longer, but it would fit perfectly, work perfectly and don't bloat on resources. Finally. It would require a life-long version updates anyway.
I share oppinion with several atheist thinkers, that life itself is such a wonder, that is no need to create additional wonder-things above that. Atheism doesn't steal wonder from things, it learns you to look for them in the very ordinary places, through knowledge. There are several laws in the universe (like the law of thermodynamics for example), that simply makes the world the way it is. You don't need testaments to obey them, you can't dissobey them. I mean can you dissobey gravity? (I mean make it non-existent, not for example compansate its effect) You can't. So if there is a question in believing that there is something bigger that us, I don't need to create gods, there is something bigger than us and we're supposed to be humble, not because of gods wrath, but because every action causes reaction and you will eventually pay for it. Now, not in afterlife. And the other thing is, gravity and other "laws" don't needs prayers, churches or groups of people who will tell other people that gravity is great. Gravity doesn't care, you just fall when stumble on Earth, that's gravity and it just works. The obvious limitation is our incomplete understanding if these phenomenons, but they work non the less. But for that reason we should be open minded as Einstein was even ybout these so called laws.

I've seen some document about various "famous" people in a past, who all over the globe shered this one thing, the feeling that there is something bigger than us and that we shall be accountable for our actions, moral etc. Curiously there were atheists, christians, buddhists, muslims, esoterically-centered etc. all kinds of people. This made me finally realise, that what is important is not a religion/or absence of religion, these are but different ways to achieve this. The document also showed many examples of people who shared same "version" of religion, but lack this.. thing..(there was a word for that but I don't know how to translate it to english).. these were the fundamentalist, extremist, blind wrong people, people hurting other people because of different beliefs, people who took the same information from each religion and took it wrong.

So, maybe we should not focus on religion at all, but on other things, is the person open-minded? Can he take facts? Can he admit he was wrong? Can he act responsibly and not arrogantly and selfish?
It's not important that he goes to the church each Sunday if he does, and it's not important that he is atheist if he's a selfish jerk who took literally "the stronger always  wins" twistedly from Darwin for a change.

Hey guys,
    Being a relatively new graduate student in a big lab where people do not have time to train me, I wanted to train myself. I read this book "At the bench" by Kathy Barker and wrote down things which were important for me. But nevertheless I thought it might be useful to somebody else also. I've attached the document with this post.
Enjoy!

I'd agree with gfischer. But if you really have to do it:
High Resolution Agarose, 0,8 - 1%
Lithium borate or Lithium acetate buffer
running distance as long as possible
not long running times (buffers allow higher voltages), e.g. 20-30V/cm
In BioTechniques (2004) 37:598-602 (Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media) you find some more informations how to do it, though you might need to do some adaptation.

leelee, on 30 August 2012 - 09:35 PM, said:

Actually that is what a dead religion is all about. Unfortunately, that probably means many religions are dead, relying on unthinking compliance to what you are told... I guess I've been fortunate to never go to church where that attitude rules.

I'm not sure that the text has to be consistent with what we understand about the universe. It was, after all, the jewish people's understanding of how everything has come t obe as it is. And how would you write the account to make it consistent with 16S data? God first made a bacteia (something invisible until the microscope was invented), then He changed it again and again and again, making bigger and bigger and more different creatures, turning some into fish, some into birds, sone into... Kinda clunky, don't you think?

Hi

There is a new electrophoresis buffer out called SB, which is just NaOH (10mM) pH adjusted to 8.5 with boric acid (works out at about 2.6g/L).   Note the lack of Tris, which is the expensive part of TBE and TAE, but works just the same, in fact you can electrophorese at much higher voltage, I routinely run gels at 200v for 0.5 hr with the same resolution and separation as 2 hours at 50v in TBE.

Check out BioTechniques 36:2 pp214-215 2004 for the article.

a friend used to use it for viscous liquids. i'm more patient.

It add to Andreea's answer, it also depends on the polymerase you used - if you used a proofreading polymerase, you can't store at 4 deg C, as the proofreading part of the polymerase can nibble away at the ends of the product.

If you are doing Realtime PCR to check Expression level. You always need to normalize your test reading with something that constitutively express in cells. Here may be GAPDH or beta- actin as constitutively expressing genes. So You have target now and GAPDH. You determine how much is expression level of your Target gene increased or decreased during experiment. Here it suggest that you have to have control where don't influence its expression level.


So here is how much your gene expressed,

Fold Change = 2^-Δ(ΔCT)

where ΔCT = CT, target - CT, GAPDH
and Δ(ΔCT) = ΔCT, stimulated - ΔCT, control

Good afternoon,

I am teacher on a high school in the Czech Republic. I teach biology and physics.
I have developed a website with recognition quizzes (plants, animals, ...), free nature photos and educational lessons. Content is still rising.
Website is non-profit. It may sounds like a cliche, but I want to help people.
My big wish is propagate this website, but I am not good enough in Internet marketing, so there are not a lot of visitors.

I would like to aks you, if you have some advice.
And I hope, that it is not a spam.

website: http://www.nablacognition.org/

Have a nice day.

With greetings
Petr Stohwasser

I'd avoid using this technique, because the usual way of pipetting is so internalised and done automatically that then I'd really have to focus what I do to avoid that the normal pipetting mode comes back. Then I'd be confused and a mixed and therefore incorrect mode would happen soon.
So only if I've to pipet glycerol what happens rarely.

use another web browser. I'd recommend firefox.

To add to Andreea's comments: It may well also be that the antibody has a non-specific band (actually this is very likely).  If you have peptide for or know the epitope of the antibody you can do peptide competition to determine if one of the bands is non-specific.

If you have cloned sequence, you could delete/mutate one of the start codons and see if you still get 2 bands.

Thank you all!
More or less was what we were thinking of. We also checked the paper where first Western was made and they used Scotch Brite sponges! Sadly, they didn't explain why.

Have a nice day!