引物设计说明-.docVIP

基因克隆 基因克隆的基本步骤: 分、切、接、转、筛、定 分:分离目标基因 (genomic DNA ,cDNA, … 切:酶切载体与克隆片段使产生粘性末端 接:连接,连接目的片段与载体 转:转化细菌 筛:抗性筛选得到阳性克隆(Amp+, Kana+, Chloramphenicol ,… 定:测序鉴定,确定正确克隆 1. ORF (Open Reading Frame /CDS(Coding Sequence Cloning primer design. The primary construction of mRNA: Procedure : 1.1 Get your sequence. 1.1.1 Go to NCBI website (/ and select the Gene database. 1.1.2 Type your target gene’s name and click search. Then select the right gene (right name and right species and click in. Here, we take NFKBIA (known as I κB α for an example. 5’-UTR 3’-UTR 5’AAAAA n —3 1.1.3 Go to the RefSeq option which contains detailed sequence information for genomic DNA, mRNA and protein of your target gene. 1.1.4 Get your sequence. There are two ways to get the ORF sequence. W ay 1: Click Consensus CDS(Here is CCDS9656.1 Then you will enter a new page. Pull down the bar then the sequences find you. W ay 2: Click NM_020529.2 to enter a new interface. Pull down the bar until you find the word “CDS”. Click “CDS”, then it will jump to the following field. The sequence in brown color is the ORF (From ATG to TAA/TAG/TGA. Congratulation you getting the sequence. 1.2 Analyzing your sequence. 1.2.1 Put your sequence into Primer premier 5.0, and then click “Enzyme” to get a new window. Select and add indicated restrict enzyme according to which vector you used for analyzing. Click “OK” when you get ready. A new window shows you the restrict enzyme sites, which should be avoided using, presented in your sequence. 1.2.2 Make a decision of which restrict enzyme to be used. Bam H1 and EcoR1 restrict enzyme sites are selected for NFKBIA ORF cloning for example. 1.3 Primers design. 1.3.1 Forward primer: Protector (about 3nt restrict enzyme siteKozark consensus sequencethe first 18-25nt starting from ATG with 1-2 C/G ending Example: NFKBIA-Bam H1-Frd: GCC ggatcc GCC ATGTTCCAGGCGGCCGAGC Forward Primer with Flag tag: NFKBIA-Flag-BamH1-Frd: GCC ggatcc GCC ATG gattacaaggatgacgacgataa