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        请教:100分的引物,到底如何

        丁香园论坛

        2031
        我初次接触PCR,准备做转基因后体内表到的半定量RT-PCR。用primer和oligo设计了一对引物,在primer上二个引物都打了100分,产物98分。但是有人说软件设计的引物有时候得分很高也会做不出来,所以想请各位高手帮忙看一下。:)

        我对这对引物有两个疑问:
        1 下游引物以AAT结尾是否不太好?
        2 产物长度是213,是否太短了,跑胶不好看?

        Selected Primers
        ------------------------------------------------------------------------------

        748U19 Upper Primer

        5' AAT GGT GGA CCG CAA CAA C 3'
        Length: 19-mer
        5' 748
        Tm: 65.7 °C (salt 1000.0 mM; oligo 100.0 pM)
        deltaG (25 °C): -37.6 kcal/mol
        Degeneracy: 1
        P.E.#: 442/442
        1/E: 5.24 nmol/A260
        31.0 μg/A260

        942L19 Lower Primer

        5' CCA AGG TAA CGC CAG GAA T 3'
        Length: 19-mer
        3' 942
        Tm: 64.7 °C (salt 1000.0 mM; oligo 100.0 pM)
        deltaG (25 °C): -38.5 kcal/mol
        Degeneracy: 1
        P.E.#: 453/453
        1/E: 5.22 nmol/A260
        30.9 μg/A260

        PCR
        ------------------------------------------------------------------------------
        Optimal Annealing Temperature: 54.5° (Max: 71.4°)

        --------------------------------------------------------------------
        Position Length Tm [°C] GC [%] P.E.#
        --------------------------------------------------------------------
        Product ----- 213 84.3 46.9 46.9
        Upper Primer 748 19 70.3 52.6 442/442
        Lower Primer 942 19 68.4 52.6 453/453
        --------------------------------------------------------------------

        Product Tm - Lower Primer Tm: 15.9
        Primers Tm difference: 1.9

        ----------------------------------
        Concentration
        ----------------------------------
        Upper Primer 200.0 nM
        Lower Primer 200.0 nM
        Monovalent Cation 50.0 mM
        Free Mg[2+] 0.7 mM
        ----------------------------------

        Total Na[+] Equivalent: 155.8

        Upper Primer Duplexes
        ------------------------------------------------------------------------------
        The most stable 3'-dimer: 2 bp, -1.3 kcal/mol
        5' AATGGTGGACCGCAACAAC 3'
        || : : ::
        3' CAACAACGCCAGGTGGTAA 5'

        The most stable dimer overall: 3 bp, -4.4 kcal/mol
        5' AATGGTGGACCGCAACAAC 3'
        ||| :::
        3' CAACAACGCCAGGTGGTAA 5'

        No stems longer than 2 bp.

        Lower Primer Duplexes
        ------------------------------------------------------------------------------
        The most stable 3'-dimer: 2 bp, -1.5 kcal/mol
        5' CCAAGGTAACGCCAGGAAT 3'
        ||
        3' TAAGGACCGCAATGGAACC 5'

        The most stable dimer overall: 2 bp, -3.6 kcal/mol
        5' CCAAGGTAACGCCAGGAAT 3'
        : || :
        3' TAAGGACCGCAATGGAACC 5'

        No stems longer than 2 bp.

        Upper-Lower Duplexes
        ------------------------------------------------------------------------------
        The most stable 3'-dimer: 2 bp, -1.3 kcal/mol
        5' AATGGTGGACCGCAACAAC 3'
        : : ||
        3' TAAGGACCGCAATGGAACC 5'

        The most stable 3'-dimer: 2 bp, -1.5 kcal/mol
        5' AATGGTGGACCGCAACAAC 3'
        || : : : :
        3' TAAGGACCGCAATGGAACC 5'

        The most stable dimer overall: 3 bp, -5.0 kcal/mol
        5' AATGGTGGACCGCAACAAC 3'
        |||
        3' TAAGGACCGCAATGGAACC 5'

        Upper Primer False Priming Sites
        ------------------------------------------------------------------------------(positive strand)
        Priming efficiency of the perfect match is 442 (above the threshold)

        Priming efficiency: 41
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        | | |||| |
        3'(506) acctctcagggcgccgacc (488)5'

        Priming efficiency: 36
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        |||||| || || |
        3'(1162) ctaccac-gggtccagtgg (1145)5'

        (negative strand)
        Priming efficiency of the perfect match is 442 (above the threshold)

        Priming efficiency: 442 (above the threshold)
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        |||||||||||||||||||
        3'(748) ttaccacctggcgttgttg (766)5'

        Priming efficiency: 118
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        | | |||||| ||
        3'(1) *tggcggagggcgtttctg (18)5'

        Priming efficiency: 77
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        || ||||| ||||||
        3'(898) ttgacacct--cgttgtgc (914)5'

        Priming efficiency: 25
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        | || || | | || |
        3'(910) tgtgcatcttgagatggtc (928)5'

        Priming efficiency: 12
        5'(748) AATGGTGGACCGCAACAAC (766)3'
        | |||||| | |
        3'(1534) gttccacctcgtcaacagg (1552)5'

        Lower Primer False Priming Sites
        ------------------------------------------------------------------------------ (positive strand)
        Priming efficiency of the perfect match is 453 (above the threshold)

        Priming efficiency: 453 (above the threshold)
        5'(960) CCAAGGTAACGCCAGGAAT (942)3'
        |||||||||||||||||||
        3'(960) ggttccattgcggtcctta (942)5'

        Priming efficiency: 63
        5'(960) CCAAGGTAACGCCAGGAAT (942)3'
        |||||| || | | ||
        3'(308) ggttcctttccatccacta (290)5'

        Priming efficiency: 34
        5'(960) CCAAGGTAACG-CCAGGAAT (942)3'
        ||| || | ||| ||
        3'(1208) ggtccccccaccggtactcc (1189)5'

        (negative strand)
        Priming efficiency of the perfect match is 453 (above the threshold)

        Priming efficiency: 81
        5'(960) CCAAGGTAACGCCAGGAAT (942)3'
        || | | | ||||||
        3'(81) gggggcgtag-ggtcctgg (98)5'

        Priming efficiency: 72
        5'(960) CCAAGGTAACGCCAGGAAT (942)3'
        | |||| ||||||
        3'(581) gattccg-agcggtcaggg (598)5'

        Priming efficiency: 45
        5'(960) CCAAGGTAACGCCAGGAAT (942)3'
        || ||||||
        3'(41) ggggaacgtgcggtgggag (59)5'

        Priming efficiency: 44
        5'(960) CCAAGGTAACGCCAGGAAT (942)3'
        || ||| || |||
        3'(1412) tgtgtcatgtcgttccagg (1430)5'

        Priming efficiency: 33
        5'(960) CCAAGGTAA-CGCCAGGAAT (942)3'
        || ||||| || | |
        3'(955) ggaaccattggccgacgact (974)5'

        附件是mRNA序列
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