Primer Premier 5 0 Load sequence Preimer Premier
- Slides: 39
Primer Premier 5. 0使用介绍 Load sequence Preimer Premier 启动界面
基本信息 Sequence name Original sequence Choose a function Use 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
引物设计界面 First you can design the primer manually Sense strand or anti-sense strand Useful information of the primer
回到主窗口 引物信息 引物及产物信息 是否出现 hairpin, dimer, false priming and cross dimer
引物编辑 Edit primer here Analysis the edit result Accept the edit result Return to the main window
Plasmid 1: Bam. HI SP NR 1 pc. DNA 3. 1 Plasmid 2 GFP
GFP Bam. HI(GGATCC) SP NR 1 -1 a pc. DNA 3. 1
NR 1的编码序列(~4000 bp) 121 ggggctccta gagaacccgg gggcgcttga ccgcggcccgcg ggtcgtacat 181 cgcgaggtcg tcgcactcgc gcaacccaga gccaggcccg 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 gtgaggacct 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 catcatcctg ctggtcagcg acgaccacga gggacgggca gcgcagaagc gcttggagac 781 gttgctggag gaacgggagt ccaaggcaga gaaggtgctg cagtttgacc caggaaccaa 红色为信号肽, 蓝色为Bam. HI酶切位点, 下划线标出酶切位点的阅读框
GFP序列(~700 bp) ATGGTGAGCAAG GGCGAGGAGC TGTTCACCGG GGTGGTGCCC ATCCTGGTCG AGCTGGACGG CGACGTAAAC GGCCACAAGT TCAGCGTGTC CGGCGAGGGCGATG 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 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 GTGACCGCCG CCGGGATCAC TCTCGGCATG GACGAGCTGT ACAAGTAA
第一步:扩增GFP基本序列 GFP序列 5’ ATGGTGAGCAAGGGCGAGGAGC …………TCTCGGCATGGACGAGCTGTACAAGTAA 3’ 3’ TACCACTCGTTCCCGCTCCTCG……………AGAGCCGTACCTGCTCGACATGTTCATT 5’ 变性, 引物复性 5’ ATGGTGAGCAAGGGCGAGGAGC …………TCTCGGCATGGACGAGCTGTACAAGTAA 3’ ………. . AGAGCCGTACCTGCTCGACATGTTCATT 5’ Primer 2 Primer 1: 5’ATGGTGAGCAAGGGCGAGGAGC………… 3’ TACCACTCGTTCCCGCTCCTCG……………CGTACCTGCTCGACATGTTCATT 5’ Primer 1: 5’ ATGGTGAGCAAGGGCGAGGAGC…………. . Primer 2: 5’ TTACTTGTACAGCTCGTCCATGCCGAGA……. Primer 1: 5’ ATGGTGAGCAAGGGCGAGGAGC…………. . Primer 2: 5’ CTTGTACAGCTCGTCCATGCCGAGA…….
第二步:GC比值;Tm值 Primer 1: 5’ ATGGTGAGCAAGGGCGAGGA…………. . Primer 2: 5’ CTTGTACAGCTCGTCCATGCCGAGA……. Primer 1: 5’ Primer 2: 5’ ATGGTGAGCAAGGGCGAGGA (GC: 60%, Tm: 64) CTTGTACAGCTCGTCCATGCC (GC: 57%, Tm: 66)
第三步:酶切位点 Bam. HI: ggatcc Primer 1: 5’ ATGGTGAGCAAGGGCGAGGA (GC: 60%, Tm: 64) Primer 2: 5’ CTTGTACAGCTCGTCCATGCC (GC: 57%, Tm: 66) Primer 1: 5’ ggatcc. ATGGTGAGCAAGGGCGAGGA Primer 2: 5’ ggatcc. CTTGTACAGCTCGTCCATGCC
第四步:阅读框 NR 1序列: atgagcaccatgcacctgctgacattcgccctgcttttttcctgctccttcgcc cgc gcg gat ccc gaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg aacagatgttccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac agctcaacgccacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt gtgaggacct…. . Primer 1: 5’ ggatcc. ATGGTGAGCAAGGGCGAGGA Primer 1: 5’ ggatcc. TATGGTGAGCAAGGGCGAGGA
NR 1序列: atgagcaccatgcacctgctgacattcgccctgcttttttcctgctccttcgcc cgc gcg gat ccc gaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg aacagatgttccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac agctcaacgccacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt gtgaggacct GFP序列 5’ ATGGTGAGCAAGGGCGAGGAGC …………TCTCGGCATGGACGAGCTGTACAAGTAA 3’ 插入GFP后的组合序列 cgc gcg gat cc. TATGGTGAGCAAGGGCGAGGAGC …………… …TCTCGGCATGGACGAGCTGTACAAG ? ? ggat ccc gaccccaag atcgtcaaca tcggcgcggt gctgagcacg cgcaagcatg aacagatgttccgcgaggca gtaaaccagg ccaataagcg acacggctct tggaagatac agctcaacgccacttctgtc acccacaagc ccaacgccat acagatggcc ctgtcagtgt gtgaggacct
Primer 2: 5’ ggatcc. CTTGTACAGCTCGTCCATGCC gg atc cct CTTGTACAGCTCGTCCATGCC
第五步 保护序列 Primer 1: 5’ GCGGggatcc. TATGGTGAGCAAGGGCGAGGA Primer 2: 5’ GCGCggatccct. CTTGTACAGCTCGTCCATGCC
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