引物设计说明-

1、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. for an exampl

2、e.5-UTR 3-UTR5AAAAA n 3' 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”. Cl

3、ick “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. Click “OK” when you get ready. A new window shows you the restrict enzyme sites, whichshould be avoided using, pr

4、esented in your sequence. Bam H1 and EcoR1 restrict enzyme sites are selected for NFKBIA ORF cloning for example.1.3 Primers design.Protector (about 3nt -restrict enzyme site-Kozark consensus sequence-the first 18-25nt starting from ATG with 1-2 C/G endingExample:NFKBIA-Bam H1-Frd: GCC ggatcc GCC AT

5、GTTCCAGGCGGCCGAGC Forward Primer with Flag tag:NFKBIA-Flag-BamH1-Frd:GCC ggatcc GCC ATG gattacaaggatgacgacgataag TTCCAGGCGGCCGAGCOrGCC ggatcc GCC ATG gattacaaggatgacgacgataag ATGTTCCAGGCGGCCGAGCThe last 18-25nt started with 1-2 C/G- restrict enzyme site-protector (about 3nt. Reverse complete this se

6、quence to gain the target primer.Example:Without flag tag: CCAGCGTCTGACGTTATGA gaattc GGCWith Flag Tag: CCAGCGTCTGACGTTA gattacaaggatgacgacgataag TGA gaattc GGC Reverse complete: GCC gaattc TCA TAACGTCAGACGCTGGSo the reverse primer is:NFKBIA-EcoR1-Reverse primer:GCC gaattc TCATAACGTCAGACGCTGGNFKBIA-

7、Flag-EcoR1-Reverse primer:GCC gaattc TCA cttatcgtcgtcatccttgtaatc TAACGTCAGACGCTGG Now you can send your sequences to companies for synthesizing.2. shRNA designThe most important thing in shRNA construction is to find efficient target sequences.2.1Find target sequence.W ay 1: Use the sequences which

8、 have been used and validated by others. The sequence in yellow frame is what you want.Here, for example, the last two TT should be removed. So the target sequence is: AGAGTCAGTTCACGGAGW ay 2: Design shRNA via RNAi designer.Take Invitrogen BLOK-iT TM RNAi Designer for example.2.2 Design your own shRNA for pSupre-puro-retro-Vector. Substitute the sequence