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        求救!引物设计

        丁香园论坛

        2309
        我设计的引物如下:
        上游引物加BamHI CGGGA TCCTGTCGCA CCAAATCGTA 73.3   62.6
        下游引物加 XbaI
        GCTCTA GACATATGCC ACCAGCAACT AACATG 70.4 65.6
        Two primers complementarity.
        Max complementarity in continuous: 4 bp, free energy= 0.20 Kcal/mol
        5'-GCTCTAGACATATGCCACCAGCAACTAACATG-3'
        ||||
        3'-ATGCTAAACCACGCTGTCCTAGGGC-5'

        Max complementarity in discontinuous: 9 bp
        5'-GCTCTAGACATATGCCACCAGCAACTAACATG-3'
        || | | ||| | |
        3'-ATGCTAAACCACGCTGTCCTAGGGC-5'



        请指点: 1引物行吗?
        2扩pcr用多少的退火温度?
        3加接头的pcr程序不一样吗?要分几步吗?
        以下是我要扩的片段!可以在前500和后700之间设计引物!那位高手帮忙一下,我要加Xba I 和BamH I 的接头!哪位高手指导一下!谢谢!
        序列:
        1 GACATGTGGA ATTTTACGCC ACCAAAAACT GCCCGCAAAA TAACTAGTTT CGCTGCTGCT ACGGAAACAG CCAAATCACA GATTCCTCTC GGGGGATGAT
        101 GGCAAAAGAA TAAAATCATT TAAGCGAAGT ATTGCCCACA AACCTCTGAA GAGACAAGAG AACCGAGGCG GTTCCAGGCT GCAAAGATAA CAGCAGCCAG
        201 GGGTTGTGAA CATGCTGGGG GCCAAACATC CCTCCCCCCC CCCTGCTCCA TCACATTTTT AATTCCCTTG CTTCGTTTCC ATAGGGGGAA GGCCAAGTTG
        301 ATTATCACCG ACCCTTTTTG GAGCCAACAA CACTTTTTCC CCAGAATTTT TGTTTTCTCT TCTTCTTCTC GCATCCCCTT TTTGTTCCCA GTCCTGTCGC
        401 ACCAAATCGT ACGGAACACT TACCACATTC GAGTATCCGT TTTGTGCAAC ACACCACCGC GCGGCCGTTC TGTTGAGCAA CGTCGGCCCG AGAGGCGAAA
        501 AAAATGAAAA CCCAAATCTA GGTAGTCAGG GTGAGGAGCA GAACGTTTGA CATCCAACCG TCTTGCTGTT AGTGAACAAG TGCCAAGATC GTAGTACCGA
        601 ATCTAGGGTA GTAGGAACAA GTAAAAAAAG GAAGCCATCA TCACAAGCCC TTGCAGTAAA GCCACATCAG CCCACCCCGT CCAAAATGAG AAAAAAGTCC
        701 CATTGAGCTG TGCCGCCGCG TCAGGCCAAA CACCAAGAAT TGGGGGGAAG CCTTTAAACT AATGCAATCG CGAAGGCAGT TTTCTTCTTC TCTTTTTCTG
        801 AGTTCCCGTT CAGCTTTGCT CCACAAAGTG GCTCTATCCC TGTTTGTCAA CGCCAGAGGC CCGGAGCGTG GTCCGGGAAT TTTTTCTCTC GCGTTCCCCA
        901 CTTTGCGCGG CAGCCATTGG AGAAGGGGGG AGGGAAGAGA AGGGGAAAAA GAAAATCTAT GACAAAACAG TGACCCACTG GCGGGGGAAG AGGAGAAGAA
        1001 AAAAGGTACC CCTCCCCAGC CTCCCTGTTT TCTTATTTTC TGGTGTTTGC TGGTCGCAAA AATTTGGGTC TCGTCGTTTG TGGTGGTGCT TTTTTTTGTT
        1101 TTTATCTTTT TTTCCCCCCT GGTTGAGGTG AGAGCCAGGT GAGTTTGGGG TCGGCCCCAT GGAGAGCGGG CAAAAATCCA GGGACTGACG AGTAGGCGTC
        1201 GATGGATGCG TGTCGTGGGT GGGGTCACCC CTACACCACC CCCCCGGTGG CAGCCCATTT TGGCTGGTGC AAACGGTGGG GGCCCTGCTT GACGGTGGAT
        1301 GATGGTCCAT CAGACGTCAA CCTTATTTGG ACATCTGCCA AGCTCAGCTT TCACTTACAC CGAGCATGGG AGAGACGCAA GGGATGGGAA TGAAGACAAC
        1401 AGAATTCCAG AAGCCGCGGA AAGACGGAAG CGAAGCATAT AATAAATGAC GGTCTGTCCT GTTCAAAAAA ACATCTATCA ATTTCCCGGT TTCCCGCCTG
        1501 CTCCATTGTT AGTGTTTCTT ACCAGGACCT GTAACCCAAT TTCTTGGGGA CCGCAAGGTA AAGAAATCAA ACTCCCCATT TTAGATTATG TCGAAGAGAA
        1601 AATTTATGGA CGTTGTTTTT CTCGCAAGAT TTTTCAGTTA TCTCTAACGA GGAGTCGCCC GCACATAGGT ACGTTTGCAT GTTAGCGTGA CCGTGCGCCT
        1701 GTCTCTCGCG CAAACAAAGA GCGAGAAGCC AGACCCACTC AAAACTGCTG CCATTGGTGC TTGCCACTTT CTATTCAGCC TTCGATCCTC TATTGAGATT
        1801 TCTATTGAGC TTCTCACTTT CTATTCAGAA TCTCATTTTC TATTCAGGTT TATTAAGTTA TTGAGGAACC AACTGGGGAC CCCACTTCGT TTCTTTATTA
        1901 ACTCGACCTT CGAACCCAGG AACCCAACTC CCTCCAAGAC TCCGACATCA CCAAAACCAC AAGACTCTTT CCATTCGATT GCGCTAGAAC TACTCACAAA
        2001 CCTCAGATTG CAAACACAAA CCCCATCGTC AGATCATTGA AACGAGGCAC ATCTACAATG GATTCATTGC AGATGGACTC CTCTCAAATG GAGGCGTTGT
        2101 CGTCGATTTG GGTCATCGTG CAGCATCAGC TCGCCCAAAT CAACAATTCC AACAACATAG CTCATATCGG CGCCGTTCTC CTCAACCAAC TTGAGGCGCC
        2201 TTTCAGGAGC ACTTTGACAT CATGGTTTCA GCAGTAAGTT CTCCATAAGC TCAAGGCTTT TCCGCAAGGG AAACTAACAT CAGGTAGGCA CACCGGCATC
        2301 AACCAGGTCT CAGTCGTGCG CGACATCAAG GCTCGTCGAC ACTACATCGG ACCAACTGAT GCTTTTCAGG AGAGCTATCA AGTTGTACAC ATCCAGGGTC
        2401 ACGGATTTGT CTGGCTTTGG GATCTCCAGG TTGATGGTGA GGATGCCAGC ATCAATTCCT GCAATATCTC TGCCGAGACA GCACAGAACT TTTCCCAGCT
        2501 GCGTCTTGAC GAGATGGCGC CTACCAATGA TGTTACGATG GTAGGTGATG AGAGCCTCAT CAACGGCAAC GGCATTGACT TTGCCATGTT GGAAGACATC
        2601 GGAAGCCCCG CATCAAACGG CTCCTCATTC CCGAGCCCTC AAGTTTTCAC CCGGGAAATT TCATTCGATG ATCTACCAGT TGGCATTATG ACTGCCGAGG
        2701 CCATTCAGAG AGACCTTCTT GGCGCCAATA TTCGCCCGGC TAACCTCATT GATCCGAGTC TTGCGAGCAA TCAGAATGTC CACAACATCA TCCATCAGAA
        2801 TAACATTGCC CAGGCTTTTA CAGATCACCT GAACATGATG GCATCCAATG CTGTCCACGA TGACACAGAA GAGGGTCTGA CAGCCGCGGA TCTTATGCCG
        2901 GCGTTGCATC GCATGTTCGT GCCGGGTCTT GAGTCAATTG CAATGGAGCA GTTCCACACC ACGCTTCCTA ATGGCGCACT CCTTCACCTG TCAATTCCTG
        3001 TCGATGAACC TGAGGATGCT GTCTCGACCA CCGCGCCTGC TGCTCAACAG ACTTCCTTGC AAGACAAGAT CCCTCGTCCT GCGAATGCCT ACATCCTGTA
        3101 CCGCAAGGAT TGGCATCCCA TAGTCAAGTC TGCAAATCCA GGAATCCATA ACAACGAAAT CTGTGAGTTC TTTTACCTCC AAACTTCCCT AGCTGCTGCT
        3201 AACTTTTTTT CCTTTAGCAA AAATCCTGGG GAAGCAGTGG GCAGCTGAGA CACCGGAAGT GCGGGCTGAG TACAAGGAGC TCGCGGAGGA GAAGAAGAGA
        3301 GAGTTCTACG CAAAGTACCC TACATATCGC TACTCTCCTC GTCGGCCGAG TGAGATAATG CGCCGAGTGA GTTTGTAACA CATTATTGCG TGAAAGAGTG
        3401 GACAGCGAGC TAACAAATTA CTTGCCAACA AGAACACGAA AAGCCGAAAG ACCCCAACGA ACGGGGCAGC GAAGCCGAGC AAAGCAAAGA AGTGCGGCAA
        3501 CGGGGTTACT GGTGGAGCAG ACAGTGCAGT CGATGTCAGC TCCCCGGGAT CGCCATTTTA GATGAGGTCA CCATTCACAT CATCCGAGAT ACATTGTACA
        3601 AGTCAGGAGC AGTTATCAAA GCTGCATTTC ACCAGAGTCC TCATATCTGA TTTTTTCTTC TTTCTTTTCA CATTGCCCCA TCGTACATCC AGAGAGCTGG
        3701 ACTCGAAATA CCCTCTCAAT AGAGTCTGCA AAGAAGATCT TAGGACCTAC GACATAGGTT ACGTGGTCAT TATTCACCAA GAACTACATA TCTGCCAGCG
        3801 AAACATATTC AGTTTTCAGA GCCATTTGTC GAAAGAAATT TTGAGATGCC GGCCCTCTGG TCACCAGTCA AAGTTTGCGG CATCGTTTCG GAGCTCAGGT
        3901 AAGCCGACCC AGAGTCCTCA CACCAGAGCG ACAGCTCTTC AGCATCTAAC GATCACAGGC TAACACTTTT GGCGCCTCAA TTAGCAAGAA CATGGCCTGC
        4001 ATGGTTTGGA TCTGGGTCGT CTGCGTTCCT CAGAAGCGGT ACCGCATCAC GACAGCTCGC TGGAACTCCA GTGGGCTGCT GGTTGGCCGC GTTGATTGTG
        4101 AGTTTTCTAC TTTCTTCACC TTTTCCTTCC GTCCCAGATC TGAGCAGAGA CTGACAAGCG ACACAAAACC TCCCCACAGC TCCTCCTCAA TCTGCCACCA
        4201 CAGCGTCCTC ACAGTCATCG TCATTCGACG GCAGTGAGGA AGACACAAAC ACAAACATCA ACGCCAATGG TATCGATGAG AGCCTGCTGC CGCCTGCTCT
        4301 CCGCGGCCGC ACTGGACCTT CCAAGCCTCC AAACCGCTGG ATTCTCTACC GTGCCGCCAA GTCAGCTGAG CTGCGGGCAG ACAACCCATC CATGAACGCG
        4401 AGCGAAATCT GTAAGTTCTT CGCTCTTTTC ACTCTTTTTC TCCGCTACCA CTTGTACCTA CCTTTTCCCC ATCTCCCATA CTCTTTGAAA AGTCTCCAGT
        4501 CCCACCTCTC AAGCTGCTGT TTTGACAAGA TAAAAGACTA ACCAACATCT CTCTCCTCAC ACAGCTCAAG TTGCATCCCT GATGTGGCAG GCAGAGTCCG
        4601 CCGCGACAAA GGCAGAGTGG GAGGAGCGCG CAGCCGAAGC CCGCGAGGAG CACATGCGAA ACACTCCAGA GTATGCGGTG GCGGCCGGTC TGCGCCAGCG
        4701 CGCCATCGCC GAGAGGAGAA GGCAGAGGCA GCAGAAGAAG AAGCAACAGG AGCAGCAGAA GGGCGACGGT GACAAGGTGG CCAAGGAGAC TTCTACATCC
        4801 ACAGAGAGCT TGGTCACTAC GGACGTCGGC GCTGTCAGCA ACCACGGCAG CAGCAGCAGT GAGGTGCCTG TCACAAGCCC ACACTCGCCG ACACTACTCA
        4901 GTCCCGCGTC TGACAGCGAT TGTACGCCCG TCGGCAGCCC TCGAGCTCGC CACGGCTGGG GTTCTGATGC TCTCAGCCTG ATGCTCGACG CCGCCGAATT
        5001 CTCTACTCTC TTCTGATCTG GTCTCTGGCA CTCTGAGCGT GCTATTCCCA GGAAATCAAA CACACAAAAA AAAAACCACA AAAAAAACTC CCCCCATTCA
        5101 CATCCATCAT ACCCCTTCGG GACTTTCCGA CATGCATATA TCTCCCACCT CTGTTTTGTT TTTTTCCTTT TTCTCCTTTG TACTTGTCAT CTTTTTTCAT
        5201 CTCAGGGTTT TGACCAGGAA CTGCCTACCT GGCTGCATAG GGACACGGCG CGATTATGAG TTTTGAGGAA ATGCTGGGGC AGCATCAGTC AGTATGCCCC
        5301 TATTTTGTTC TCAAAGTTTA CTTGTTCTGT TTTTAGCTGG TTCTTCTGCA TCATTCACGG GAGAAGGGGA ATAAAGGAGT CTTCACAGGC ATATGACCCA
        5401 TCATGACATA GATACCCTTA CGTTTTTCTT TTCTTTTCTT TTCTCAAGTC TCTCTGTTCT TTTACAGTTG CGCATCAGAT GGGATGACAC AAGATGTTAC
        5501 CACGAGAAAT GCACTCAGCA AGTCGATACA CTAAGTCAAT CATTCGACCT GTTACCAACT CTAAAAACTC CTCGCTACGT GCCATTGTTT GTCCTAGTGA
        5601 AACTGCCGAT GAGATATGTG TGATATGAGA TTACAATGGT GACAATGGTA ACAAGATGTG TCGTCCGCCC TGTAGCATAG TTTGCTTTGA CGTAGAAACC
        5701 CAACGCCAGT AGCGGCACTC CGCCGCCTTA GGTCTGCTGT GACGCGTTCC AGTCGCTGCT CCAGATGAAT GTGCCGCATC TCCACTCGGT ACCTTTTTCC
        5801 TTCTCGCCTG ACGGCCCAAG AGGTCTGCCG CAAATGTAAA AGGACCGGCC TGCATGACAA GAAGGTAAAC ATGTTAGTTG CTGGTGGCAT ATGTCACGAA
        5901 GAGCCGTTTT AATACGTTGC TATCAAGATG CTGTCTCAAG CGGGCCACGA GGCGGCAAGC ATACAAGCTT GCATAAAGTA CAGACGATGA ATGAGGCAGA
        6001 TAAAGACTAG GAAAATACGA GAAGAAACAT TTTCTGTCAG ACATACCACA ATTGATGCCT GGCTTCTTGG TCTGTAGGCT GATGCACGGT TCGTCGTGCT
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