引物设计教程

1、PCRPreimer Premier 启动界面Load sequenceSequence nameOriginal sequenceUse these two button to translate the DNA seq to a protein seq or a protein seq to a DAN seq 8种密码子偏好Choose a function First you can design the primer manuallySense strand or anti-sense strandUseful information of the primer引物类型搜索模式5引物

2、位置范围3引物位置范围产物大小范围引物长度28对引物引物分值100分为满分每对引物的信息双击选中一对引物回到主窗口引物及产物信息是否出现hairpin,dimer,false priming and cross dimer一对理想的引物应当不存在任何一种上述结构，因此最好的情况是最下面的分析栏没有But Edit primer hereAnalysis the edit resultAccept the edit result Return to the main windowPer 25-merPer 25-merPer 5-merOpen sequence fileG Internal S

3、tabilityFrq为邻近6至7 个碱基组成的亚单位在一个指定数据库文件中的出现频率。该频率高则可增加错误引发的可能性。上游引物下游引物只是示意图Key information of primerHairpinDimer and cross DimerGC%Total PCR informationSearch primerPreimer Premier 启动界面Sequence nameOriginal sequenceUse these two button to translate the DNA seq to a protein seq or a protein seq to a D

4、AN seq 8种密码子偏好Choose a function First you can design the primer manuallySense strand or anti-sense strandUseful information of the primer引物类型搜索模式5引物位置范围3引物位置范围产物大小范围引物长度28对引物引物分值100分为满分每对引物的信息双击选中一对引物回到主窗口引物及产物信息是否出现hairpin,dimer,false priming and cross dimer一对理想的引物应当不存在任何一种上述结构，因此最好的情况是最下面的分析栏没有But

5、 Edit primer hereAnalysis the edit resultAccept the edit result Return to the main window任务用绿色荧光蛋白(GFP)标记蛋白NR1举例说明SPBamHIpcDNA3.1NR1Plasmid 1:Plasmid 2GFPSPBamHI(GGATCC)pcDNA3.1NR1-1aGFP 121 ggggctccta gagaacccgg gggcgcttga ccgcgcgcgg gcggcccgcg ggtcgtacat 181 cgcgaggtcg tcgcactcgc gcaacccaga gccagg

6、cccg ctgtgcccgg agctcatgag 241 caccatgcac ctgctgacat tcgccctgct tttttcctgc tccttcgcc c gcgcggatcc 301 cgaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg aacagatgtt 361 ccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac agctcaacgc 421 cacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt gtg

7、aggacct 481 catctctagc caggtctacg ctatcctagt tagccacccg cctactccca acgaccactt 541 cactcccacc cctgtctcct acacagctgg cttctacaga atccctgtcc tgggactgac 601 tacccgaatg tccatctact ctgacaagag tatccacctg agtttccttc gcacggtgcc 661 gccctactcc caccagtcca gcgtctggtt tgagatgatg cgagtctaca actggaacca 721 catcatcc

8、tg ctggtcagcg acgaccacga gggacgggca gcgcagaagc gcttggagac 781 gttgctggag gaacgggagt ccaaggcaga gaaggtgctg cagtttgacc caggaaccaa NR1的编码序列(4000bp)红色为信号肽, 蓝色为BamHI酶切位点, 下划线标出酶切位点的阅读框 ATGGTGAGCAAG GGCGAGGAGC TGTTCACCGG GGTGGTGCCC ATCCTGGTCG AGCTGGACGG CGACGTAAAC GGCCACAAGT TCAGCGTGTC CGGCGAGGGC GAGGGCGA

9、TG CCACCTACGG CAAGCTGACC CTGAAGTTCA TCTGCACCAC CGGCAAGCTG CCCGTGCCCT GGCCCACCCT CGTGACCACC TTCGGCTACG GCCTGCAGTG CTTCGCCCGC TACCCCGACC ACATGAAGCA GCACGACTTC TTCAAGTCCG CCATGCCCGA AGGCTACGTC CAGGAGCGCA CCATCTTCTT CAAGGACGAC GGCAACTACA AGACCCGCGC CGAGGTGAAG TTCGAGGGCG ACACCCTGGT GAACCGCATC GAGCTGAAGG

10、GCATCGACTT CAAGGAGGAC GGCAACATCC TGGGGCACAA GCTGGAGTAC AACTACAACA GCCACAACGT CTATATCATG GCCGACAAGC AGAAGAACGG CATCAAGGTG AACTTCAAGA TCCGCCACAA CATCGAGGAC GGCAGCGTGC AGCTCGCCGA CCACTACCAG CAGAACACCC CCATCGGCGA CGGCCCCGTG CTGCTGCCTAC CAGTCCGCCC TGAGCAAAGA CCCCAACGAG AAGCGCGATC ACATGGTCCT GCTGGAGTTC GT

11、GACCGCCG CCGGGATCAC TCTCGGCATG GACGAGCTGT ACAAGTAAGFP序列(700bp)5 ATGGTGAGCAAGGGCGAGGAGC TCTCGGCATGGACGAGCTGTACAAGTAA 3 3 TACCACTCGTTCCCGCTCCTCGAGAGCCGTACCTGCTCGACATGTTCATT 5GFP序列5 ATGGTGAGCAAGGGCGAGGAGC TCTCGGCATGGACGAGCTGTACAAGTAA 3 3 TACCACTCGTTCCCGCTCCTCGCGTACCTGCTCGACATGTTCATT 5Primer1: 5ATGGTGAG

12、CAAGGGCGAGGAGC.AGAGCCGTACCTGCTCGACATGTTCATT 5 Primer2Primer1: 5 ATGGTGAGCAAGGGCGAGGAGC.Primer2: 5 TTACTTGTACAGCTCGTCCATGCCGAGA.Primer1: 5 ATGGTGAGCAAGGGCGAGGAGC.Primer2: 5 CTTGTACAGCTCGTCCATGCCGAGA.第一步：扩增GFP基本序列变性, 引物复性Primer1: 5 ATGGTGAGCAAGGGCGAGGA.Primer2: 5 CTTGTACAGCTCGTCCATGCCGAGA.Primer1: 5 A

13、TGGTGAGCAAGGGCGAGGA （GC:60%,Tm：64）Primer2: 5 CTTGTACAGCTCGTCCATGCC （GC:57%,Tm：66）Primer1: 5 ATGGTGAGCAAGGGCGAGGA （GC:60%,Tm：64）Primer2: 5 CTTGTACAGCTCGTCCATGCC （GC:57%,Tm：66）BamHI: ggatccPrimer1: 5 ggatccATGGTGAGCAAGGGCGAGGA Primer2: 5 ggatccCTTGTACAGCTCGTCCATGCC 第四步：阅读框atgagcaccatgcacctgctgacattcgc

14、cctgcttttttcctgctccttcgcc cgc gcg gat ccc gaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg aacagatgttccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac agctcaacgccacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt gtgaggacct.Primer1: 5 ggatccATGGTGAGCAAGGGCGAGGANR1序列：Primer1: 5 ggatccTAT

15、GGTGAGCAAGGGCGAGGAatgagcaccatgcacctgctgacattcgccctgcttttttcctgctccttcgcc cgc gcg gat ccc gaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg aacagatgttccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac agctcaacgccacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt gtgaggacctNR1序列：5 ATGGTGAGCAAGGGCGAGGAGC TCTCGGCATGGACGAGCTGTACAAGTAA 3 GFP序列cgc gcg gat ccTATGGTGAGCAAGGGCGAGGAGC TCTCGGCATGGACGAGCTGTACAAG ?ggat ccc gaccccaag atcgtcaaca tcggcgcggt gctgagcac