1 首先一定要确保“Exon junction span”选项设置为“primer must span an exon-exon junction”,这是为了排除DNA污染。2 产物长度一般设置为90-180为较好。3 Tm值要在60℃左右,且正反引物的值要接近,不能差太远。4 GC含量也要接近,不能差太多,一般在45%-55%之间较好。5 自我配对不能太高。6 越靠前...
荧光定量PCR中,为了消除基因组DNA对实验的影响,引物设计时一般会选择跨内含子;值得注意的是,如果对引物是否跨内含子有要求,必须选择序列登录号的方式导入基因序列,程序才能自动识别外显子的拼接位点。 “No preference”代表无要求,“Primer must span an exon-exon junction”代表引物必须跨越跨内含子,“Primer may ...
首先一定要确保“Exon junction span”选项设置为“primer must span an exon-exon junction”,这是为了排除DNA污染。Tm值要在60℃左右,且正反引物的值要接近,不能差太远。
4 点击进入"Primer-BLAST"5 从第二步的页面复制序列或者序列号,粘贴在第一个框里。修改产物长度,即“PCR product size”,一般为100-200之间最好。还要选择“Exon junction span”为“primer must span an exon-exon junction”,这是为了排除DNA污染。其它的参数都可以不改。6 把网页往下拉到底,点击左下角的...
如果你想设计的引物是跨内含子和外显子的交接处,在这里可以设置成primer must span an exon-exon junction。外显子的匹配碱基数和内含子的长度等设置一般选择默认。 (4)特异性(Specificity) 在specificity check区,选择设计引物或验证引物时的目...
如果你想设计的引物是跨内含子和外显子的交接处,在这里可以设置成primer must span an exon-exon junction。外显子的匹配碱基数和内含子的长度等设置一般选择默认。 (4)特异性(Specificity) 在specificity check区,选择设计引物或验证引物时的目标数据库和物种。这一步是比较重要的。这里提供了7种数据库: RefSeq ...
To avoid possible genomic DNA amplification, the option "Primer must span an exon-exon junction" is selected. As shown in Figure 3, Primer-BLAST successfully returned five specific primer pairs and the detailed alignment between targets and pri- mers are shown. During the search process, Primer...
第一步:新建序列(或者通过Open直接打开序列) 第二步:确定你要设计引物的起始位点,双击下面的序列可以选中一段(暂时先不要管引物长度) 第三步:选择作为上游引物Select->Forward Primer,然后你会发现下面的序列上有相关引物。首先一定要确保“Exon junction span”选项设置为“primer must s...
Exon/intron selection To avoid amplification of contaminating genomic DNA, design primers or probes so that one half hybridizes to the 3′ end of one exon and the other half to the 5′ end of the adjacent exon. To do this, simply select “Primer must span an exon-exon junction”. You...
such primers can successfully distinguish between cDNA and genomic DNA. When possible (i.e., when a genomic sequence with one or more intron(s) is available), primers that span an exon-exon junction are favored, especially when the junction occurs at the 3' end of the primer, to further...