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2

I'm gonna throw a curve ball here. :) I think the "housekeeping" or "reference gene" concept is fundamentally flawed, because it rests on circular logic. In order to verify that a "housekeeping" gene X is constant across your samples, you must assume that the $C_t$ values themselves are actually a valid measure, so that you can assess the expression level of ...


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As pointed out by others a reference gene should not change its expression between the control and test samples. Reference gene need not be a housekeeping gene; housekeeping genes are generally used because they are present in all cells are are not subject to any specific regulation. However, global changes in the cell, such as stress or development, can ...


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It may depend on which protocol you looking at, but you can use a specific primer set for the first PCR and another specific primer set for the second PCR. Even if you use a none specific primer set for the first PCR, it could be easier to amplify your target sequence because your target sequence would be more abundant than the original solution before the ...


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200uM dNTPs in 50ul reaction mix could produce more than 10ug DNA if all dNTPs were consumed whatever the length of PCR products is. You could detect as low as 5ng DNA fragments by conventional electrophoresis using EtBr. You probably could see a clear band when you load more than 100ng of a DNA fragment. Therefore I am not worried much about the dNTP ...


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Extracting genomic DNA and PCR are different processes. Extracting genomic DNA means that you get all the DNA sequences from human genome. Using extracted DNA, you can amplify a specific region from the whole genomic sequence. A primer set is usually set at the both ends of a sequence you want to amplify the sequence. In your case, you the region ...


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The References and External Links sections of the Wikipedia article in your question contain a great deal of relevant information to get you started. There is a link to the FBI's CODIS and NDIS (National DNA Index System) Fact Sheet, which contains a lot of relevant information, including part numbers for validated kits from several manufacturers. DOI ...


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If you're looking for reference sequences, RefSeq is a good bet. http://www.ncbi.nlm.nih.gov/nuccore/?term=18S+ribosomal+RNA+AND+srcdb_refseq%5BPROP%5D


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You need to watch out though. Some very used genes could be very uninformative as housekeeping genes in qPCR. ACTB and GAPDH have tons of pseudogenes (analogous genes not yielding proteins) that can interfere with the PCR. You can read more in this article (1). To make it even worse, the PCR amplification fragments can be of the exact same length as the ...


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Yes, it does matter. Gene expression varies a lot thoughout tissues and living conditions, so you need to test a variety of genes and choose the ones with the smallest variation in the expression between sample and control. If you don't find any, you have to test other genes for this purpose. This is important, as this can severly affect the outcome of ...


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I have used both Gibson assembly (what you mean by assembly?) and LCR as replacements for blunt or sticky ended cloning. LCR has, for me, worked better in that a higher proportion of constructs that come out have been assembled correctly and I have noticed no mutations after LCR while I have seen them somewhat often with Gibson.



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