Bluenoise

Okay I've figured it out. It's pretty easy now. just zoomed through 9 and 10. btw after 10 when the circles touch they just start overlapping at the start. I've got to run away.

Level 7 was the first to take some time. But now I got to goto lab. :/ It's not too difficult actually if you just make lots of little shapes. I like it but it gets annoying how you have a limmited space to work in. It's hard squeezing those fat circles in sometimes.

Oh I see what you mean. I guess no matter how you look at it some bias is unavoidable if you want to generate fragements that small. But I think my purposes can tolerate a little DNase I bias. The genome that I'm fragmenting is only a few megabases in size so even within 1ug I should have tens of billions of copies of it. And I only need a few of every 300bp section represented. So It will be inevitable that I will get that even with a little bias. I want to avoid RENs since no matter how you look at it some sequences will just not contain a cut site within 1kb no matter which ren you use.

Fairy tales are for children. They may even have some nice moral lessons in them. But they're still fairy tales. Ha! 20 words.

Martin I think we should maybe open an new amazon Bestseller list analysis section for you. But yeah I don't really think I have any one comment on this. Hmm except maybe it's occuring because women read more novels and men tend to focus on the big popular non-fiction works. Thus you'd expect women to write more novels and men to write more non-fiction works. Now the novels would have the audience much more fragmented than the current big group of non-fiction works. For various reasons. Novels don't get outdated to the same degree, and poeple have much more individual preferences, etc... In other words you have a large group of men focusing on a small amount of books writen by men. And you have a larger group of women deviding their attention between a large group of books mostly writen by women. Thus that may likely explain the observence uhh maybe I did have a comment on this.

CviJ1 sounds interesting. It would be easier to control and produce all blunt ends, but it would definatley have a much higher sequence dependence as well. If your using DNase I in blunt ending conditions (Mn2+, followed by filling of small overhangs). Then wouldn't every site be a site adjacent to a pyrimidine, since there is always a pyrimidine on one of two strands at every site? And thus have no dependence on this? My biggest concern is calibrating the DNase I reacton environment to get semi-reproducible fragmentation.

Like localized to an area of your skin, or the underlying nerves? Because that is the only area you could hope to localize it too. Past that it would just enter the bloods stream and be distributed.

The large amount of ionizable groups makes it very water soluable. However it still may not be able to pass through the skin. Why would you want to apply it in a cream anyways? Seems like a really ineffective and wasteful way of adminitering it. It's a nutrient, and takes a while to get to it's destination, so it's not like applying it dermally will prolong the effect. It's fairly slow acting. Would probably be much more effective in pill form. I'm fairly skepticle that you wouldn't get the same effect just from typtophan itself.

Hmmm I don't think I'd agree with that. DNase will produce a random library, as it has no bias to sequence, as long as the DNA is completely purified from associated proteins. Other enzymes will not ie RENS. I'm sorry you're just going to have to trust me that I need a library of 300-500 bp inserts. 1kb is for to big for my purposes.

I'd say the best way to measure is to bend your fingers 90 degrees to your palm and measure from the back of the nuckle. But yeah what is the accepted way to measure ones finger. Because I get very different ratios when trying different methods.

To tell you the truth I like windows. It never causes me any problems. I'm always pretty skeptical when I hear out people complaining about problems with it. Cuz I've used it for a long time and never had much issue. Maybe I've just learned to use it in such a way that doesn't encourage these things to happen? Plus I work in research and well many of these software apps just aren't there for mac or linux. Or are way to expensive (especially for macs). Plus I have software at school that only works on windows. Mac OS is annoying since it doesn't allow me to do things the way I want. I wont argue this point. Cuz this is just personal preference. I just really don't like how it's layed out. Linux I loved when I used it. But well it's just for to much work setting it up. I have far to busy of a life to deal with that. It has even been difficult for me to find much time to post on this board. I've had to cut out alot of things from my life recently and well that's one of them. Though if I find the time I will go back to using linux.

Are you just doing this by observing your hand? Because if you look at your hand your index will look longer since it sticks out farther. That's what I thought when I looked at mine. However once I take a ruler and meansure from the base of the finger on the palm side to the tip the ring finger is obviously longer.

Uhh they were referring to how much you could get back from the blood...

That's was excellent. Most things were really recognisible. Anyone know the link so I could try to dowload it permenantly? The .swf just links to a video somewhere... Though in a real cell things are much more congested and not quite so orderly. That wouldn't really make for much of a video. I like how they use the actual structural images from .pdb to render the proteins. Very realistic. Much better than the typical spheres and ovoids.

I've had this problem twice before when having duel boot linux and windows. Those two kids just don't get along sometimes. Never had any problem with windows by itself. Never really tried linux by itself since windows is an absolute necessity for me.

Wouldn't this also apply force to the tapered walls of the cavity?

Well you can't control what it cuts. But all reactions have a rate associated with them. So you just need to stop it before it's complete, and the DNA will be partially cut. The mean length proportional to the reation time.

Okay I know in single straded form DNA of 7 nucleotides has been shown to circulize. However, dsDNA is like a rigid rod. Most natural plasmids are at the smallest 1.5- 2 kb long. I've been told that a dsDNA sequene of around 600bp would thus be very unlikely to circulize in a ligation reaction. Which would leave this said ligation of long linear chains forming. Some of which would circulize in the end when they got long enough to favor it. Anyone know on the size limit for the circulization of dsDNA in a ligation reaction?

Well I'm basically looking for short DNA sequences that exhibit certain properties. I have desinged a method to assay for this and capture said sequences. Besides that I really can't really go into it in more detail sorry. The organism is a simple bacterium. (I know pretty vauge but I'm held under disclosure conditions so I hope you understand) Well I'm partial to the DNAse treatment since it will not have a bias towards sequences like the random primers or restriction digest would. It is completely non-specific in where it cleaves DNA. Thus I know that if I have a pool with a high enough concentration of fragements they will almost surely cover the genome a few times over in overlapping fragments. I'm afraid if I have a bias towards certain sequences I will loose what I'm looking for. Controlling for size is fairly simple though. First I'd Need to calibrate the DNase reaction to determine the reaction conditions where the mean length of cleavage products falls within the range I'm looking for. After that I just run the cleavage products on a gel and cut out and purify the fragments that run within that size class I desire. Say between 300-500 bp.

Sorry I don't know about in the UK. But in Canada, where the law is probably really simular. You're alowed around 12 kilos of ore before you need a permit. I recently became a certified nuclear energy worker . Which means my lab can dose me with more radiation then the general populace without breaking the law .

Well decided to go with the DNase treatment. If anyone has a comment don't be too afraid to reply.

This sounds incredibly painful and horrible. But if you're into that maybe some tabasco sause would be safer... capsasins are pretty harmless.

I have a friend who works as an arborist. He spends all day swinging around in trees like a chimp except with ropes and chain saws. Conseqently he's incredibly ripped. He also seems to have lost alot of deterity with his hands. He now writes little better then how you'd expect a chimp too. Maybe there's some truth to this.

Wow you sound like some PETA guy. You do realised how hard animals have it in the wild? You do realises it's not Disney fantasy world where they hold hands and dance around in circles? When an animal is in the wild it is generally in a constant strugle to survive. While a pet is garunteed shelter, food, and companionship. No mater how stupidly it acts. Why do you think bears that are fed by people generally become dependant and try to move into human civilization. Because it's a hell of a lot more dependable than relying on nature to provide it.

I'm curious as to what ya'll think is the most effective way of taking genomic DNA and chopping it into random fragments of a defined size. Say around 500bp. I'd think ultrasonification but 500 bp is probably to small for that. So maybe a time calibrated Dnase treatment or even a restriction digest. There's also pcr with random primers. But which of these methods would be the best to represent the whole genome in Random 500bp fragments?