Hot answers tagged pcr
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All sequencing methods, be it classical Sanger sequencing or next-generation sequencing (or even third generation) need a certain amount of DNA to work with.
You either need to extract DNA from a large-ish tissue sample or you need to amplify DNA content from a smaller sample. The first approach is often impractical, or downright impossible (when you want ...
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Your teacher is indeed correct.
In the first round you would get two identical molecules of the dsDNA.
In the second round you would get 3 identical molecules and one molecular with an A substituted for a G in one of the strands. ie.
No error (3 of the 4 molecules):
------G-------
------C-------
One mismatch (1 of the 4 molecules):
------A-------
...
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Perhaps you can draw inspiration from classic paper on lambda cloning: Maniatis T, Hardison RC, Lacy E, Lauer J, O’Connell C, Quon D, Sim GK, Efstratiadis A. 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15: 687–701.
Try selecting tissues from the animal which you think is "enriched" (i.e. highly expressed) for the specific ...
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Deletions may make sense if you are analyzing the N-terminus or C-terminus of a protein. If you are looking at an internal region however, keep in mind that the more AAs you delete, the more likely you are to disrupt the overall protein structure. If you delete any random selection of 8 AAs within a protein, there's a chance you'll knock out activity by ...
7
I will format it to .ppt as soon as I have more time! If something is not readable, please let me know!
I have to point out several things:
The fraction of incorrect DNA molecules does not depend on the number of cycles (as long as the number of cycles is higher than 2).
There is one exception: if the mutation occurs outside of the region to be amplified ...
6
While it isn't the cheapest, it is certainly the fastest and simplest. I would quikchange out the amino acid. This would require no subcloning and only require
two ~25 nt primers ($10)
1 shot of pfu (~$0.25)
1 shot of DPNI ($0.05)
competent cells (~$5)
sequencing to confirm (~$4-6)
Overall, probably >$20 a mutant all in 2-3 days of waiting.
(edit) I'm ...
6
I see big fuzzy bands around 100bp as well. They're most likely RNA contamination. To get rid of them, digest your RT-PCR products with RNAse-H. But if you just need to visualize your band of interest, and the fuzzy bands aren't getting in the way, it shouldn't be a problem.
I usually input anywhere from 1-2 ug of RNA into my RT-PCR reaction using the ...
6
Primer Tm calculations can vary significantly based on the method used. What I can tell you, is that the Tm really depend on the polymerase you are using for the PCR reaction and for each polymerase there is a set of PCR conditions you have to follow. As I use Phusion® Hot Start High-Fidelity DNA Polymerase from Finnzymes, I will give you an example with ...
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Western Blot tests on young children are practically useless, since they test for antibodies. The child will likely have antibodies passed down by the HIV+ mother, regardless of whether the child has HIV. The test will show the antibodies, which may be mistaken for an active immune response from the child. As such, there will be a high false-positive rate ...
4
This paper describes some PCR strategies with LINE and SINE PCR identification (Shedlock and Okada. SINE Insertions: powerful tools for molecular systematics. BioEssays (2000) 22:148-160.).
I have no experience with PCR amplification of SINEs or LINEs, however I can think of two strategies right now.
1) You may be able to find a unique 18-20 nt region ...
4
We do it all the time. You can use one of your end/flanking primers and use that as a primer for sequencing. Companies will typically have a setup where they can take a "premixed" sample.
Since we tend to use sequetech, here are their details: http://www.sequetech.com/requirements.php?premixed=1
For a second opinion look at Elimbio's: ...
4
I don't think primer dimers are your primary concern here. Usually in my experiences, I get primer dimers all the time, even if the reaction works and I get my bands of interest.
Maybe you ought to troubleshoot other aspects of your PCR that might account for why your reaction isn't working. Have you tried using a positive control with your primers? You may ...
4
There is no single "ideal amount" of RNA. I would suggest you to do a titration curve to determine the best amount for your specific assay. The band at 100 bp could be a non-specific amplification. You could try to increase slightly the annealing temp to see if that removes (or reduces) the lower band. Alternatively, you can consider a different primer set ...
4
According to their website New England Biolabs use a version of the approach pioneered by Wayne Barnes, as described in:
Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147
This is basically an assay for the mutation rate in a PCR-amplified lacZ (β-galactosidase) gene, assayed by transforming E. coli, plating on the ...
3
Everything depends upon the infection and on the general immune status of the patient.
Generally, the prerequisite for DNA to freely circulate in the blood is the presence of bacteria themselves in the blood (bacteraemia). This means that the infection left its original site (where it is usually kept isolated from the blood flow by the immune system). ...
3
Please provide more information: fold-change relative to what?
If you did what I think you did (single control gene that you calculated fold change to of your gene of interest) I'd say this is the wrong approach. What you need is a set of genes which have similar expression levels across all your samples (controls and cases) to be able to compare your gene ...
2
In my own personal lab experience, I got some unexpected results using Phusion polymerase. However, I have not seen deletions, only insertions (ranging from single bases to 3 tandem copies of a primer sequence). This is not an answer to your deletion question, but it does suggest unusual things might happen.
I have seen deletions with T4 DNA polymerase ...
2
You also probobably need to check if your samples haven't been contaminated with PCR reaction inhibitors, which is very common if you first extract your mRNA, digest remaining DNA and then run a PCR with a less then 10-fold dilution. You need at least a 200 times dilution to get rid of all the artifacts.
Once you've diluted your samples, place a repeats of ...
2
The thermal cycler adjusts sample temperature based on the volume of the sample. So if you run samples of different volumes side by side, not all of them will cycle through the optimal temperatures of each step. If you were working with a "hot start" polymerase this is even more critical, as the Taq won't be able to amplify at all unless the sample is heated ...
2
I haven't done ARMS PCR myself, so my answer is based on theory and there might be other practical factors I overlooked.
The characteristic feature of the Vent exo(-) polymerase is the lack of exonuclease activity compared to the regular Vent polymerase. ARMS uses the fact that a mismatch at the 3' position prevents primer extension. For this to work it is ...
2
It is important visualize more faces to design primer or oligo and PCR experiments:
- calculate thermodynamic parameters of DNA Hybridization (Oligo/Template)
compute hairpin-loop, dimer, bases penality and melting temperature about primer or pair primer
calculate statistics about melting temperature with the change in composition and mix PCR ...
2
I am not sure of a case where Taq doesn't add 3' A overhangs.
You could use TA cloning to clone your PCR product. The basic principle is to use a vector with 3' T overhangs. If you have a vector without these overhangs, you can use a terminal transferase + dTTPs to create them (or even Taq, but this is not as great because it adds a purification step to ...
2
Reverse transcriptase (and most other commercial enzymes these days) is made recombinantly, then purified to varying degrees, using varying methods, among different suppliers (and sometimes between different lots from the same supplier). Some enzymes actually have multiple functional units combined in a complex, others may require a post-translation ...
2
RNAses are enzymes, and there are various ways to inactivate them. Unfortunately, RNAses are rather stable proteins and autoclaving doesn't completely kill their activity. The common methods to inactivate are unspecific methods that will destroy any enzymes, either through covalent modification or degradation.
The most common methods for RNAse inactivation ...
1
You have to design your primers properly. Usually, in real-time PCR, you don't choose a very long product. Ideal product size is 150-300.
Next, see what your NRTI is analogous to. For e.g. if I am using AZT (Azathymidie), I would place my reverse primer at or after the last T.
There are alternate techniques as well. You can use primer extension ...
1
What kind of reverse transcriptase is used in qRT-pcr is there a standard like taq dna pol is used for qPCR
M-MuLV (Monoley murine leukemia virus) reverse transcriptase
Some RT enzymes are engineered to remove the RNAseH domain so that template RNA persists for more rounds of RT.
Is it possible to use a biologically active Telemorease Elongation ...
1
Judging by your post history, it seems like you may want to pick up an introductory book on molecular biology. This question is really asking a number of questions. I will attempt to answer the difference between PCR and RACE, and I would suggest asking the others as separate questions, as @dd3 has suggested.
Polymerase Chain Reaction (PCR) is the basic ...
1
The one that actually makes me skeptical of the use of this is that this "cure" was primarily a massive dose of antiretrovirals used as catchup therapy because the infant's mother didn't have access to prophylactic treatments while she was pregnant and delivering.
Basically, it's a cure that requires you to have access to antivirals very, very near the ...
1
Look at chemical hydrogen bond breakers. Guanadine hydrochloride is included in the buffer conventionally to compete with the the hydrogen bonds that form the double helix. Other hydrogen bonders like Propionamide and 2-Pyrrolidone can weaken the helix and I would think lower the melting point of the bonds.
How much the Tm is affected would depend on the ...
1
Perhaps isothermal amplification is possible (NASBA)?
Amplification of DNA also seems possible (NASBA at biomerieux).
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