How to analyze PCR and RFLPs, and how does the interpretation change when you kno the actual sequence?
91642
- with PCR you amplify a DNA sequence (like ATGGTAGCATGATC etc etc etc)\n- P = polymerase (thats the name of the enzyme that does the amplification\n- C = chain (there are actually two chains: first the chain of nucleotides ATGGAGACCCTAetc and second the chain of polymerization reactions)\n- R = the enzymatic amplification reaction\n- you can only do a PCR with a DNA double strand, because you need two starter oligonucleotides that bind to the initial template that you want to amplify; they resemble a short DNA sequence of the complementary (opposite) strand; they work best when they have a length between 18 and 20 nucleotides. When you do a PCR, you will most likely know the sequence of these oligonucleotides and you will order them from a company who designs them for you (you just give them the sequence you desire and they make them fo you)\n- so when you do a PCR, you have to offer the polymerase enzyme some material so that it can work: i.e. the template DNA that you want amplified, the starter oligonucleotides and especially the single nucleotides A, T, G, C\n\n- for RFLP you will need an enzyme that cuts the DNA strand\n- restrictions enzymes cut specific sites\n- they have names like BamHI EcoRI etc etc etc\n- example: BamHI cuts at the site GGATCC and only there\n- so when you have a DNA sequence that is, say 2000 basepairs long, and you have only one GGATCC, the enzyme will cut only once. If the DNA sequence was linear (i.e. it has two ends like a sausage) then you will get 2 DNA fragments. If your DNA is circular, you will get one fragment\n- so think in larger dimensions: if you cut a tremendously long DNA sequnece, you will have more GGATCC sites, consequently you will get more fragments.\n- so if you know your sequence, you can just do your simple math calculations and predict the fragments and their lengths: from the fist GGATCC to the second its, for example 10,010 basepairs, from the second to the third xxx and so on\n\n- R = restriction (cutting the DNA strand into pieces)\n- F = fragment (the pieces of DNA that arise when cutting the large one)\n- L = length (the different lentgs of DNA pieces that arise during the cutting)\n- P = polymorphism (referring to the fragment pattern when comparing them to the pattern of different samples)\n- the actual RFLP comparisons are made in the gel, because sometimes you don't have the exact DNA sequence of every DNA you want to analyze\n- for example, you cut the genomic DNA of two or more related bacterial species. And lets say you will know the exact sequence of only one of them. For that one you can make predictions about fragment lengths based on your calculations. For the others you can't, because maybe they have mutations in their sequences.\n- so when you compare the fragments to each other and to the one you initially precalculated, you might find that the other related species reveal different fragments. That means the sequences af the repated species have alterations in the site where the restriction enzyme was supposed to cut.\n- So the interpretation will be that specific DNA sites have an altered sequence to the sequence that was given to you\n- you can also make predictions about total mutations in the DNA, if you know how long the total DNA was and if you know how many restrictions sites there are and how many were not cut with a specific enzyme
852174
Sounds like a typo.  Call the lab that performed the test.
827602
You're doing the PCR reaction to look for an amplification of a DNA segment.  When you run it out on a gel and look for the presence of bands of DNA, water will be your negative control because there is no DNA.  You want to do this to have something to compare your amplified sequence with; you also want to do this to make sure that bands in your amplified lanes are due to PCR and not to something DNA that was present in the water.
730267
Mass-spectrometry (MS) is the only technique that gives true quantitative answers with extremely high certainty, but MS of DNA is uncommon and hard to do.  A more typical approach would be to "run a gel" separate the different fragments based on size: you have a porous matrix, you place the mixture of DNA pieces on one end, you apply a voltage.  Since the DNA is charged it migrates on the gel, but the bigger pieces are hindered by the matrix (the gel).  You then run a "standard" side-by-side with your unknown mixture--something with a mixture of a few fragments on known size.  Typically these "standard ladders" which are commercially available come in increments, e.g. 100 bp, 500 bp, 1000 bp, etc.  So you will be able to tell the size is between those fragment lengths (e.g. that a band from your unknown mixture is between 100 and 500 bp) with very high certainty, but you do not know the exact length with any certainty.\n\nAlternatively you can sequence DNA--this is very easy and cheap now-a-days, and has a very high-certainty.  So after doing a gel you can cut out the different bands and have them sequenced individually.  Both steps--the gel and the sequencing--are very routine, cheap, and reliable.
1020699
First of all make sure your template is good quality. Did you extract genomic DNA, do you have a library in plasmids...(?).\nTry re-purifying your template.\n\nMaybe your template is GC-rich or has some secondary structure.\nIs your template very long?If so make sure you let the polymerase is working at the elongating temperature for enough time (1min/kb)\n\nIf you are using plasmid template try not to use too much. Don't put more than 30-40ng DNA in a reaction. \n\nCheck your primers:\n-can they form a dimer?\n-can they anneal at more than one site on the template DNA with a only few mismatches?\n-is the difference in the Tm of the two primers more than 6 C (it shouldn't;ideally they should be the same and I try not to exceed a difference of 4)?\n-do your primers end in more than 3  c or g in a row?\n-what's their Tm and GC content?Tm should not be very low (I design my primers at 56-61) and GC 40-60%.\n\nThe most strigent conditions are using as annealing temperature 2 C below the lowest Tm of the two primers. Try lower temperatures.\n\nYou can try different enzymes Taq, Pfu, Pwo, etc, even mixtures. Not all enzymes have the same processivity.\n\nYou can try different Mg+2 concentrations.\n\nThere are a lot of control experiments you can do depending on your template and primers. Eg if you have a library in a  plasmid that has sites for universal primers you can use one such primer and the other respective primer you designed in order to check if there is something wrong with the quality of your primers. \n\nIf everything is perfectly designed and you've tried evreything but still get a very faint (but of correct size) band, gel purify it and use it as a template for a new PCR. In this case you'll have a very high chance of getting mutations so you'll need to use a high fidelity enzyme like Pwo or Pfu turbo and of course sequence a lot till you get a correct clone.
773115
you can surely rule out some things based on the type of plate you are using.  assuming that you have covered that base, you can separately pick a colony that you think may be a contaminant and look for differences in growth patterns, color, morphology, gram stain, growth on other media, and if all else fails, you can sequence it to find out exactly what it is.
393032
1. Isolate the genome of organism on which the desired gene sits.\n2. Cut it with restriction enzyme with the desired recognition site.\n3. Perform polymerase chain reaction procedure with oligomer primers which have the suspected complimentary sequence with your gene\n4. Perform agarose gel electophoresis to isolate the multiplied sequences\n5. Separate the duplex structure of primer:DNA in alkaline medium.\n6. Perform Southern Blot Transfer (could be found in any molecular biology laboratory manual)\n7. Transform the target organism with the resulting gene fragments from the southern blot. (transformation procedure vary with the varying target organism)
771405
40.\nhttp://www.research.att.com/~njas/sequences/b000602.txt
116081
OK.  I've searched all of my micro stuff and I can't find mention of a PCR anywhere.  You'd figure someone who could answer this question would know what a PCR was, but not this someone.\n\nAll I can say is that for years people relied on bacterial morphology (Spherical, Rods, Curved, Clusters, Chains, etc. . .), grams stain characteristics (+ or -), colony morphology, atmospheric growing conditions (O2, no O2, increased CO2), nutritional requirements, and biochemical tests (Peroxidase, Indole, Oxidase) to differentiate different species of bacteria from each other.\n\nI hope this is what you're looking for.
273434
There are multiple start codons, depending on the species.  But I'm pretty sure that there are always a few oddities in how different species might encode information is slightly non-standard ways.
79133
It is used to quantify how much of a mRNA transcript is in a sample. RT is for reverse transcriptase, which is used to make a cDNA (complimentary DNA) copy from the mRNA (messenger RNA) template. DNA is stable, whereas RNA is not. The cDNA can then be subjected to PCR using the primers of your choice to amplify your transcript of interest.\n\nThe point is that this tells you how actively a gene is being transcribed in a given cell under given conditions.
732538
1RR, 2 Rr, and 1 rr   (a genotypic ratio of 1:2:1)\n\nwe just learned this in my bio class too!\nyou can use a punnett square to solve problems like this. you can look up punnett squares online, but you would set one up with an R and an r on both sides.\n\ni hope that helps!
157565
RT stands for Reverse Transcription, which refers to the key difference between the two. In PCR the DNA is heated so the strands separate, starting sections called primers attach, and then polymerase finishes out the strand, duplicating the amount of a particular section of DNA. This is then repeated many times, until you have millions of one (or more) sections.\n\nWith RT-PCR, you start out with RNA instead of DNA. Reverse transcriptase is then used to build DNA from the RNA, which is then duplicated through the process above.
532414
Unless you get someone to confess, your chances of proving the test was altered are pretty slim.  Do you believ there is actually ZERO chance (not just "hardly any" chance) your best bet is to get the test repeated by an independent laboratory.  Get new samples and send them to another lab.  Even better, get enough for two swabs, and send each set to different labs.  \n\nNeedless to say, this is going to cost some money, although it is less than 18 years of child support.  So you ought to be *sure* before you try to challenge the test results.  "Sure" does not mean just "well, I was careful."  It certainly doesn't mean wrong time of the month.  Sure is more like, "I was out of town for the entire month and never saw her."  \n\nAnd I should warn you that even if you get a negative test result, that just means there are two different results, and a court might choose to believe the other one.  That's one reason to take two additional samples.  Only you know if what the odds are that any independent labs will come to a different result than the first one.
103798
When DNA fingerprinting first began, restriction fragment length polymorphism (RFLP) analysis was used, though it has been almost completely replaced with newer techniques. RFLP analysis is performed by using a restriction enzyme to cut the DNA into fragments which are separated into bands during agarose gel electrophoresis. Next, the bands of DNA are transferred via a technique called Southern blotting from the agarose gel to a nylon membrane. This is treated with a radioactively-labelled DNA probe which binds to certain specific DNA sequences on the membrane. The excess DNA probe is then washed off. An X-ray film placed next to the nylon membrane detects the radioactive pattern. This film is then developed to make a visible pattern of bands called DNA fingerprinting. By using multiple probes targeting various polymorphisms in successive X-ray images, a fairly high degree of discrimination was possible. The primary drawback of RFLP is that the exact sizes of the bands are unknown and comparison to a molecular weight ladder is done in a purely qualitative manner. Many labs developed policies that described what they considered a unique band, but it was not standardized and led to DNA fingerprinting coming under harsh attack in People v. Castro 545 N.Y.S. 2d. 985 (Sup. Ct. 1989). RFLP was a very time consuming method which required relatively high quantity of good quality DNA to be used (such as a dime sized blood drop). This made typing degraded samples such as those from evidence that had been exposed to the elements fairly difficult.
838778
To be honest with you i don't quite understand your question, it is often that restriction enzymes are used to create genomic library for which you look into the overlapping sequence and then be able to determine which fragment is next to which. If that is what you have done, you should have used the fragmented DNA sequence to determine the orginall DNA sequence that you digested it with the enzyme, to confirm the sequence you ahve found is correct, you can use PCR, you got the hypothetical sequence, thus by making primers from the start and the end of the sequence (doesn't matter which way round) you should have amplify the same sequence which you started with, you got the amplified DNA (same as the orginal DNA because you used primers that complemented the very start and the very end of the sequence which you found out after the restruction enzyme mapping) run it on gel electrophoresis with a DNA size marker that will include the size of DNA you have sequenced, if the amplified DNA size is the same as the sequence you have found from restriction mapping, that means your primers did complemented from the start and the end, that means the sequence you thought is actually the correct sequnece.\nI am sorry, i don't think i am making this clear enough, but anyway, that's the best i can think of.
931137
It really depends on the lab.  In my lab, they extract DNA and run markers to find what genes the animal has.  In other labs, you may find the actual sequence of the DNA (A-C-T-G).  Genetics is a large field and no two labs do the same thing.