/sci/ - Science & Math

>>5265039

What about it?

>>5265088
How exactly does it go about obtaining its readings? My solution had labeled and unlabeled dCTP. How do i determine the ratio of the two?

>>5265168
It basically keeps replicating a desired sequence, and counts how many of the fragments that are the sequence.
I think.
Haven't reached that part of The Molecular Biology of the Cell 5th ed.

>>5265314
Ok, so the control well had no polymerase. That's to see how many fragments the initial solution had without the replication process?
How does the pcr indicate/detect that the amplicon (replicated fragment) was made?

>>5265336
I think they use chips with nucleotide bases on them,
that light up when bound to.

>>5265341
I see. Is that where the dCTP (Deoxycytidine triphosphate) come in? Some of it is labeled which maybe was used to see if the nucleotide was used in the replication of new fragments?
But some of it was not labeled. How would one determine the fraction of labeled nucleotides in the amplicon?

>>5265373
Well you know how many are labeled and how many aren't, couldn't you just take the ratio of labeled CTP and unlabeled CTP and apply it to the pcr?

>>5265391
I meant the ratio after being taken up and used to make the fragments. Or in other words the newly made fragments are composed of both labeled and unlabeled nucleotides; what is that ratio?

>>5265437
Of the L and UL CTP in each fragment?

>>5265458
Yeah. Or maybe not each fragment but the total L and UL dCTP in all fragments might work.

>>5265489
It would be the same as the original ratio wouldn't it?
What other supply of cytosine would be present to change that ratio?

>>5265540
I have been assuming that not all of the nucleotide was used up. If so the labeled could have been used more or less than unlabeled, shifting the original ratio.

Should I assume all of the original reactants are used?

>>5265555
I don't see why not.

>>5265567
Ok nice. That makes things a bit easier.

I still have some things I need to figure out, but you've been a great help. Thanks for taking the time to help me out.

I FUCKING HATE THOSE PCR TUBES. I have to cut them with the fucking scalpel when I don't need to do 8 rxns. And if you want to spin them by sticking them into a 1.5ml tube then get sucked in. I despise those shit but some idiot bought a whole bag of them.

Quickly ask specific questions if you have them. I am a professional PCR'er.

>>5265592
No problem, but again, I'm not certain on any of this.

>>5265631
You should probably trust this guy more.

>>5265631
Ok heres a few:

Some alternatives to PCR for making a probe that could use dCTP?
If trying to amplify a dozen or so molecules of DNA to microgram amounts, what are some precautions to take?

>>5265662
>Some alternatives to PCR for making a probe that could use dCTP?

Not sure what you mean here, you want to attach something to dCTP that you can later detect in a synthesized fragment of DNA? To incorporate an NTP such as CTP you will need to use a polymerase of some sort, depending on if you are starting with RNA or DNA template. You don't necessarily have to make it a 'chain reaction' so you don't amplify your fragment, but just make one strand. For example going from RNA->DNA just making one strand you incubate your dNTPs (with dCTP replaced with a labeled dCTP) and a reverse transcriptase at like 50C for about an hour. This is stuff you do for sequencing really, can't come up with a good reason to do it otherwise, maybe detect where your DNA travels. AKA incorporate a labeled NTP, transfect into cell/virus and study where it goes. So that would be like fluorescent labeling and using the fluorescent microscope. Regardless, it sounds to me like you are talking about pyrosequencing which you can wiki.

>If trying to amplify a dozen or so molecules of DNA to microgram amounts, what are some precautions to take?

Seems pretty standard? What you could do is run your standard PCR, then PCR purify out your amplicon and run PCR again. Can't think of any 'special' precautions I would take in this case relative to any other.

>>5265715
Ok, I'll look up pyrosequencing on wiki.

How does the pcr indicate that the amplicon was made and what is the mechanism for this detection?

Thanks for the answers so far.

>>5265731
For your standard PCR you take your product and run it out on an agarose (typically) gel. The fragments you amplified separate on this gel by size. So let's say you want to check if your cells are expressing Gene X. You design primers for this gene, lyse the cells, extract its DNA, run a PCR to amplify that little chunk of geneX for which you have specific primers, and then run the amplified product on a gel. You can see the DNA on the gel by a couple of different techniques. The principle is the same: something binds just the DNA so you can see it. Generic method is to add ethidium bromide to your agarose, run your DNA on it, then check it under a certain wavelength UV light where the EthBR intercalates between the DNA strands and fluoresces as a distinct band.

There are other more complicated ways of detecting that are more quantitative rather than just checking for the presence of something. This is called qPCR. You can detect the concentration of your starting sample by using fluorescent probes that are detected by a sensitive machine in REAL TIME (so the more product you make, the more of the probe you bind, the more you fluorescence you detect). How you actually get numbers out of this method is a little involved. But I can direct you to some protocols/manuals.

>>5265787
The PCR was run as a real time PCR. It was also asymmetric, if that means something.

I'm less concerned about how the machine gets the numbers as much as the mechanism for the probes. My understanding is it is a hairpin strand with the fluorophore and a quencher, and when bound it stretches and then the fluorophore and quencher are far far apart enough for a fluorescent signal to be put out.

>>5265815
Oh, so you want the complicated stuff lol. There are two probes I know of: the taqman probe and the SYBRgreen. SYBR is not specific to your sequence it will bind to any double stranded DNA (therefore pick up primer dimers, but then again with SYBR you don't have to design a bloody probe and you can check for dimers with the melt curve).

Taqman probe: is what I think you are talking about. Essentially you design a sequence complementary to the target sequence. On the 5' and 3' end of this sequence you have your fluorophore and quencher. The flurophore fluoresces at a certain wavelength and because the quencher is so close it absorbs that radiation and converts it into some other non-fluorescent form of energy. When the polymerase gets in there and starts to displace the probe, the fluorophore gets far enough away from the quencher to show detectable fluorescence. Wiki has a diagram under "taqman probe".

>>5265815
Oh and the asymmetric part: they used less of one primer to create more of the strand complementary to the taqman probe. This is because as you create more amplified fragments they tend to anneal to each other preventing the probe from binding and giving inaccurate results in later cycles of the reaction.

>>5265831
Oh no. Trying to figure this on my own I read up on what I described but didn't realize it was Taqman. I'm not sure what the probe is but I do know it specifically mentions it not being the normal high temperature Taqman and that the PCR is instead running at 20 degrees C anneal temperature. So I am using the SYBRgreen then?

>>5265851
Holy jesus 20 degrees? I can't imagine why you would do that. I don't even know if that would work. You usually decrease annealing temp if you are trying really hard to make some very rare template to amplify. But it also increases nonspecific amplification.

Taqman probes are designed usually to have melting temp of high than your primers, and SYBR has nothing to do with temperature really it just binds stuff.

If you tell me more about whatever the experiment is/was maybe I can figure out what they were trying to do.

>>5265851
Now that I think about it, if it was asymmetric it is most likely taqman, doing asymmetric with sybr would be counterproductive.

The annealing temp is stupid low though, 45 is like reaching, below that I've never even thought about.

>>5265864
I have it written here that the low temperature is due to a short template.

This is actually supposed to be part of an overall course experiment in a lab to help me learn PCR and other techniques. In the end we want to synthesize and purify a deoxyribonucleotide analog labeled with fluorescent dye and demonstrate its ability to be used for making a probe by PCR.

>>5265887
Annealing temp is nothing to do with length of template it has everything to do with the melting temp of your primers so they anneal specifically to your intended gene sequence.

Short template: decrease elongation time.

Sounds like a neat exp. I think there are a few things like this that exist, they use something like it I think for some version of pyrosequencing.

>>5265908
Ok, man. Thanks for all your help, you're a real cool dude. I'm going to head out now. You've been a great help in figuring this out.