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No about 16,000 bases is about the limit that you can do for PCR so perhaps you could use an entire viral genome (or a cDNA copy) as a PCR template but not an entire genome of cellular organism even if you ignore the fact that a eukaryotic organism has it's genome spread over separate chromosomes.
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∙ 14y ago
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What is the defference between Real-time PCR and reverse transcriptase PCR?
Difference between real time PCR and reverse transcription PCR is as follows:- 1. Real time PCR is donated as qPCR and on the other hand reverse transcription PCR is denoted as RT-PCR. 2. In qPCR, the template used is single strand DNA strand whereas in the RT-PCR, the template used in process is single strand of RNA. 3. The real time PCR enables both quantification as well as detection of the DNA in the real time whereas the RT-PCR enables only the quantification of the RNA and it is little bit slower process then the qPCR as it first produce the cDNA from the template RNA strand and then process it in the similar fashion as the traditional PCR.
What causes smear instead of a clear band in PCR?
A smear in your PCR can be caused by too low annealing temperature. Too much or little magnesium or poor primer design or most likely poorly extracted DNA for your template. Precipitate your template DNA again with 70% ethanol give it a good dry and re-elute and try once more. You can also try diluting your template. If there are PCR inhibitors in your DNA prep then using less template can improve results. If you have plenty of DNA in your prep then a 10 or even 100 fold dilution shouldn't prevent good amplification.
What is pcr and its application?
PCR or polymerase chain reaction is a method to amplify a fragment of DNA. PCR reaction contains template DNA, primers, dNTPs, polymerase enzyme, buffer and water. The thermocycler manage the heat and time to synthesize DNA (denaturation, annealing and extension). The main application is one can amplify the gene or DNA of interest to millions of copies by using this prior to cloning.
Why do you resolve your PCR products by electrophoresis gel?
PCR products produce million copies of your gene of interest. After PCR, we usually resolve them on the agarose gel to visualize the amplified DNA using EtBr stain under UV. The main purpose is, it make sure your gene is really amplified and the length it run is corresponding to the right size of your gene of interest and purify it from other template DNA and other unspecifically amplified DNA products by extracting from the gel.
What are the pros and cons of RT-PCR?
There are several advantages of using real-time PCR over other methods. Real-time PCR assays are thousand folds more sensitive than RNase protection assays or dot blot hybridization. It allows you to quite precisely calculate and compare of the amount of template in each cycle, instead of determining the amount of product at the end of the reaction. Quantitative RT-PCR is commonly used for clinical applications. For example, you could use this method to quantify the amount of HIV RNA particles per ml of blood plasma in a patient who is undergoing treatment with antiviral drugs to see if it's working or not. The main problem with real-time PCR is that it requires specialised thermal cyclers (PCR machine) with fluorescence monitors and its reagents are quite expensive.
How does a DNA molecule act as a template?
Template DNA is a DNA you want to amplify. So you should know what you are amplifying before a PCR or you can make it by sequencing your PCR product.
What is the sequence of the template DNA?
What do you really want to ask? template DNA is a DNA you want to amplify. So you should know what you are amplifying before a PCR or you can make it by sequencing your PCR product.
What is the defference between Real-time PCR and reverse transcriptase PCR?
Difference between real time PCR and reverse transcription PCR is as follows:- 1. Real time PCR is donated as qPCR and on the other hand reverse transcription PCR is denoted as RT-PCR. 2. In qPCR, the template used is single strand DNA strand whereas in the RT-PCR, the template used in process is single strand of RNA. 3. The real time PCR enables both quantification as well as detection of the DNA in the real time whereas the RT-PCR enables only the quantification of the RNA and it is little bit slower process then the qPCR as it first produce the cDNA from the template RNA strand and then process it in the similar fashion as the traditional PCR.
What causes smear instead of a clear band in PCR?
A smear in your PCR can be caused by too low annealing temperature. Too much or little magnesium or poor primer design or most likely poorly extracted DNA for your template. Precipitate your template DNA again with 70% ethanol give it a good dry and re-elute and try once more. You can also try diluting your template. If there are PCR inhibitors in your DNA prep then using less template can improve results. If you have plenty of DNA in your prep then a 10 or even 100 fold dilution shouldn't prevent good amplification.
Why initial denaturation step is done before denaturation step in PCR?
To make sure the double-strand DNA template is separated into single strands.
What are the different types of polymerase chain reaction techniques?
types of pcr: AFLP -PCR. Allele-specific PCR. Alu-PCR. Assembly -PCR. Assemetric -PCR. Colony -PCR. Helicase dependent amplification. Hot start pCR. Inverse -PCR. Insitu -pCR. ISSR-PCR. RT-PCR(REVERSE TARNSCRIPTASE). REAL TIME -PCR
What organism is used primarily in PCR technique?
well in PCR technique it is a part of DNA whcih is used to polymerized or multiplied but for studing Genetic engineering we generally prefer most studied microorganism i.e. E.coli
What is primer in pcr?
A primer (oligonucleotide of a specific sequence) is required for Taq polymerase to extend the template strand by adding complementary nucleotides. The function of the primer is to anneal to the template strand at a very specific site and facilitate the initiation of strand elongation mediated by Taqploymerase.
What is pcr and its application?
PCR or polymerase chain reaction is a method to amplify a fragment of DNA. PCR reaction contains template DNA, primers, dNTPs, polymerase enzyme, buffer and water. The thermocycler manage the heat and time to synthesize DNA (denaturation, annealing and extension). The main application is one can amplify the gene or DNA of interest to millions of copies by using this prior to cloning.
Does PCR require knowledge of the DNA sequences at the ends of the region to be amplified?
Yes, the primers need to anneal at the correct sites on the template strand for the specific region to be amplified. For the primers to attach to a specific site, they need to be in the correct sequence -- one that is opposite to the template sequence.
Role of additives in PCR?
Reactants: (dNTPs, template DNA (to be amplified), primers(bind to DNA to begin elongation of strand), DNA Polymerase (elongate DNA), & MgCl2) in buffer + H2O
What is pcr and types of pcr?
PCR is a biotechnological method to amplify your gene (DNA) of your interest. It produce millions of your DNA fragments hence used in cloning. There are variants of this method using the same thermocycling principle such as touch down PCR, gradient PCR, RFLP, multiplex PCR, Q PCR, RT PCR and so on.
