rt-pcr引物设计原则和方法（RT-PCR primer design principles and methods）.docVIP

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rt-pcr引物设计原则和方法（RT-PCR primer design principles and methods）.doc

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rt-pcr引物设计原则和方法（RT-PCR primer design principles and methods）

rt-pcr引物设计原则和方法（RT-PCR primer design principles and methods） RT-PCR primer design principles and methods Search the NCBI for the gene, find the mRNA of the gene, locate the coding region in the CDS option, and in the origin below, the Copy encodes the sequence as a candidate for the software query sequence. Open Primer Premier5, click File-New-DNA sequence, appear the input sequence window, Copy target sequence in the input box (select As), in this window, the sequence can also be directly translated into protein. Click Primer to enter the primer window. This window can be linked to a primer and Edit primer and search results option, click the Search button to enter the primer search box, select PCR primers, Pairs, set the search area and the length of primers and length of products. In the Search Parameters, you can set the corresponding parameters. In general, if there is no special needs, parameter selection default can be, but the product length can be appropriately changed, because 100 ~ 200bp product electrophoresis run more scattered, so you can choose 300 ~ 500bp. Click OK to start the automatic search software as a primer, the search is completed, will automatically jump out of the window, the default search results according to the score (Rating) ranking, click on any of the search results, in the primer window, showing the overall situation of the sequence and position of primers, including upstream primer and downstream primer the primer, all kinds of information. For primer sequences, can simply check, avoid the following situations: 3 end dont end with A, it is G or C, T can also do not appear; 3 3 consecutive bases connected, such as GGG or CCC, otherwise easily lead to mismatch. This window should focus on: Tm should be between 55~70 degrees, GC% should be between 45% and 55%, the upstream primers and downstream primers Tm value is best not to differ too much, probably below 2 degrees better. The bottom two of the window lists two levels of structure