难道连primer premier 也没有办法吗？
丁香园论坛
659
真郁闷啊，一个目的基因扩增了3个月也没出来，换了好几个引物，都是文献上的，结果不是产物bp数不对，就是目的条带超暗还有非特。
一咬牙，决定赶鸭子上架自己设计一把，结果用primer premier 5.0设计出来的引物，一个90分以上的都没有，也没有错配、发夹等全“NONE”的结果出现，拿分值最高的去OLIGO验证，实在很不理想。当然我知道不可全信软件，但是总得找个让自己信服的根据吧。唉，老板天天对我叹气，说你什么时候能给我个结果啊，我找谁叹气去啊~~~~~~~~
基因的全序列我贴在下面，恳请主任们大侠们不吝赐教，真的不胜感谢！
cggagccgtc cccgacgcca cctcccgtct cctccgccgc agtttcctcc gccgctgtcg
61 ggggtgcaga gctgaggggc tcgggcagcg cgccgcgcag ctcctctcca gtcgcccgcc
121 tcgccgtcct ccccaccgag ggctcgcccg ggcgcccctc ctcagccgcg ccgggtctcc
181 tcgcgcagcg ccgcggcctg cgctacctgc ggccggctgg caacagaggc aacgcacact
241 cgcggcgggg cgggaggctc gcagcccctc gggcgggctc ggcgaggccc ggccgcgtgc
301 ggcggtttct gggggagggg gctcgggtga ttcagcgccc ggtcaggcgg aagcggccgc
361 cgccgcggag gaggaggaga aagaggaggg gacagctgtc cgcgcccggg cgcccgcggt
421 ggcacgagcc cgcggcccga gtgcgaggcg gaggcgagga ggccgcgggg acgcgaggcg
481 aggccggccg gggcccccgc agccatggag aacgctcaca caaagaccgt ggaggaggtg
541 ctgggccact tcggcgtcaa cgagagcacg gggctgagcc tggagcaggt caagaagctc
601 aaggagagat ggggctccaa cgaattgccg gctgaagaag gaaagacctt gctggaactt
661 gtgatcgagc agtttgaaga cttactagtt agaattttat tgctggcagc atgtatatct
721 ttcgttttgg cttggttcga agaaggtgaa gaaacgatta cagcctttgt agaacctttt
781 gtaattctgc ttatattggt agccaatgca attgtgggtg tatggcagga gagaaacgct
841 gaaaatgcaa tagaagctct taaggagtat gaacctgaaa tgggcaaggt gtatcgacag
901 gacagaaaga gtgtgcagcg gattaaagcg aaagatatag ttcctgggga tatagtggaa
961 attgctgttg gtgacaaagt tccggctgac attagattga catccatcaa gtctacaact
1021 ctgagagttg accagtcgat tcttacaggt gaatctgtct cggtcatcaa gcatactgac
1081 cccgtccctg acccacgagc tgttaatcaa gacaaaaaga acatgctctt ttctggcaca
1141 aacatcgctg ctggcaaagc tatgggagtg gtggtggcga ctggagtcaa tactgagatc
1201 ggcaagatcc gggatgaaat ggttgcaaca gaacaggaga gaacacccct acagcagaag
1261 ctggacgagt ttggggaaca gctttccaaa gttatctccc tcatttgcat tgcagtctgg
1321 atcatcaaca tcgggcattt caatgaccca gttcatggtg gctcttggat cagaggtgcc
1381 atctactact ttaagattgc agtggccctg gctgttgctg ccatccctga gggtctgcct
1441 gctgtcatca ccacctgctt ggctcttgga actcgaagga tggcaaagaa aaatgctatt
1501 gttcgaagtc tgccttctgt ggaaaccctt ggttgtactt ctgttatctg ctcagacaag
1561 accggcacac ttaccacaaa ccagatgtcc gtctgcagga tgttcattct ggacaaagta
1621 gaaggtgata cttgttccct taatgagttt accataactg gatcaaccta tgcacccatt
1681 ggagaagtgc aaaaagatga caagccagtg aaatgccatc agtatgacgg gcttgtagag
1741 ttagcaacga tctgtgctct gtgtaatgac tctgctttgg attacaatga ggcgaagggt
1801 gtgtatgaaa aagttggaga agccacagag actgctctca cgtgcctggt agagaagatg
1861 aatgtattcg acacggagct gaagggactt tctaaaatag aacgcgccaa cgcctgcaac
1921 tcggtcataa aacagcttat gaagaaggaa ttcacgctag agttttcacg tgatagaaaa
1981 tcaatgtccg tctactgtac accaaacaaa ccgagccgga cgtccatgag caagatgttt
2041 gtgaagggtg ctccagaagg tgtcatcgac aggtgcaccc acatccgagt tggaagtacc
2101 aaggtcccca tgacgcctgg tgttaaacag aagattatgt ctgtcattcg ggagtggggc
2161 agtggcagcg acacactgcg gtgcctggct ctggccactc atgacaaccc gctgaggaga
2221 gaggagatgc acctggaaga ttctgcgaac ttcatcaaat atgagaccaa tctgactttc
2281 gttggctgtg tgggcatgct ggaccctccc aggattgaag tggcctcttc tgtgaagctg
2341 tgccggcaag cgggcatccg agtcatcatg atcactgggg ataacaaagg cactgctgtg
2401 gccatctgtc gccgcattgg catctttggg caggatgagg atgtgacatc aaaggctttt
2461 acagggcgag aatttgatga attaagcccc tcagcccaga gagacgcctg cttaaatgcc
2521 cgttgttttg ctcgagttga accttcccac aagtctaaga tcgttgagtt cctgcagtcc
2581 tttgatgaga tcacagctat gactggtgat ggtgtgaacg acgcgcccgc tctgaagaag
2641 tcggaaatcg ggattgccat gggctcaggg actgcagtgg ctaagacggc ctctgagatg
2701 gtgctggccg acgacaactt ctccaccatc gtggccgctg ttgaggaggg gcgcgccatc
2761 tacaacaaca tgaagcagtt catccgctac ctcatctcct ccaacgtggg ggaggtggtc
2821 tgtatcttcc tgacggcagc ccttgggttt cctgaggctt tgattcctgt ccagttactc
2881 tgggtcaatc tggtgacgga tggtctgcct gccactgcgc tggggttcaa tcctccagat
2941 ctggacatca tgaacaaacc cccacggaac ccaaaagaac cgctgatcag cgggtggctc
3001 tttttccgtt acctggctat tggctgttat gttggcgctg ccaccgtggg tgctgctgcg
3061 tggtggttca tcgctgctga cggtggtccg agagtctcct tctaccagct gagtcatttc
3121 ctgcagtgta aggaggacaa cccagacttc gaaggagtgg attgtgcaat ctttgagtcc
3181 ccgtatccga tgacaatggc actttctgtt ctagtcacca tagagatgtg caatgccctc
3241 aacagcttgt ctgaaaacca gtccctgctg aggatgcccc cctgggagaa catctggctc
3301 gtgggctcca tctgcttgtc catgtccctt cacttcttga tcctctacgt ggaacctttg
3361 ccactcattt tccagatcac accgctgaat ctgacccagt ggctgatggt gctgaaaatc
3421 tccctgcctg tgatcctcat ggacgagacg ctcaagtttg tggcccgaaa ctacctggag
3481 cctggtaaag agtgtgcgca gcctgccacc aaaccctcct gctccctgtc ggcatgcacc
3541 gacggcattt cctggccctt tgtgctgctc attatgcccc tggtggtctg ggtctacagc
3601 acagacacta actttagtga tatgttctgg tcttgactga cagcgctaca tgcagaagat
3661 gtttaactta atcaattaat tttttattgt ttaaagcaac tgtctatttc tgctgaattt
3721 tcacatgaac atattggctg gtgaaggagg tttcatatct agattttgtt ttgctttttc
3781 tgactccagt gggacaagat tttccttttt tttatacaca taattaaagt gtccattgac
3841 atgtacagag aactaacact attttatgca gatatttttt tgtagatgaa aaaagcatgt
3901 acagtgttct gtttcatact catacttgta caaagatcgt tgagccagca gacattgtca
3961 gcaagttaat tggcagcaga cttaggaaat gaatgtgtgt ggtttttaaa agaaaaaaaa
4021 ctaaatagca tgtattgtgt cttttgcatg attctctgga tttaatttgg tatcacagtc
4081 taatttttat tcataagcca atttttctgc actgagcaga gtcctgctac ctcagtcagt
4141 atttttggtt tgccactccc tgcaccctca gcctctgtca cccccagccc accctggccc
4201 gctcggctgc ttctgtaggt ctgatggttc tgtttacatc tgtcttcacg aggtttgcct
4261 gtctcgcttg tgcagaaaac attgctccag atccaatcga ctgggtttct gtcccttcgc
4321 ctagttttta aggttattta tttaaatgtc taatgtattt tattgtaaca gacattgttt
4381 tgccaacatt gcctattcag tggcacttca caatctagtt taaaagaaaa ataaaacatt
4441 ttaaatggac agaggaaaat aactgtcttg ttcttaatgg ccttgaacta cccatgatca
4501 gttcaaattc ctgagcttct cagctcggag ccttagtttc ctgttcctga gaagaacgtg
4561 tactgctgca cagtagggat tatctataat gtacaggcac gatcgtcatg tgacaaacat
4621 ttactcaagt ttctgctggc ctggtgtatg taggctggtg taagggcaag tgtgtgtgtg
4681 tatgtgtgtg tgtgttctgt aaaatctgta aatagcacat gaccaaatga actgtataga
4741 aatattttta tttgaatatg gcactaacca ccaccaccat gactgaccgg acgtttgcgc
4801 atgttcaagg tcagtcttaa cggcagtgtg agtcattaac agtcctaact gtggtgtttt
4861 cctccaatgc cttccaacat ccatcaacta accaatactg gagtaaccgc ttcctaaaca
4921 ttgcagaaat gtaagggtgt tcgggtgcgt gcatgtgcgt tgttagcaac acatcttcca
4981 gccctctgca tgactgagct tggggaaaga gaaatagaac agcccccagc tcactgtgtg
5041 atgtggagga aatgtgtatt acaagtgggg ttttagctgt tgagtcaaaa taataacaag
5101 tgtacaattt agcataagga atcggagagc ctctccagag aagtcggttt ctttgctgca
5161 agaagaatga ggttctgaac ccttatccaa gaacagaagc catcagccaa gtctccacat
5221 ttctctgcaa aatgttgtag cctctataac tgtatgatag tgtaatgcat gccttcagtt
5281 gtaagtggcc agatcgcgct acagtgacat tgaaacctgc tctctaattg gccctgtaca
5341 gtttgcttat ttataaattc atttaaaaca ctacagctgt tgaatggtta caacctaggc
5401 ctccggtcct aacttcagtt gttctcctcg gtgtgcagcc agctgttcca cactgtatta
5461 ttgtaactta tttagtgaag tcagaagcag tagacagatg ttggtgcaat acaagtattg
5521 tgtgcattta tcgtaataaa gtgctccgcg tcggttcagt tcctcacagc ttctcacagt
5581 gcatgtctga ctgtagtctg taaatagagg tcagtgtccg tgctgctaac aggtatcgat
5641 cgcacaga
一咬牙，决定赶鸭子上架自己设计一把，结果用primer premier 5.0设计出来的引物，一个90分以上的都没有，也没有错配、发夹等全“NONE”的结果出现，拿分值最高的去OLIGO验证，实在很不理想。当然我知道不可全信软件，但是总得找个让自己信服的根据吧。唉，老板天天对我叹气，说你什么时候能给我个结果啊，我找谁叹气去啊~~~~~~~~
基因的全序列我贴在下面，恳请主任们大侠们不吝赐教，真的不胜感谢！
cggagccgtc cccgacgcca cctcccgtct cctccgccgc agtttcctcc gccgctgtcg
61 ggggtgcaga gctgaggggc tcgggcagcg cgccgcgcag ctcctctcca gtcgcccgcc
121 tcgccgtcct ccccaccgag ggctcgcccg ggcgcccctc ctcagccgcg ccgggtctcc
181 tcgcgcagcg ccgcggcctg cgctacctgc ggccggctgg caacagaggc aacgcacact
241 cgcggcgggg cgggaggctc gcagcccctc gggcgggctc ggcgaggccc ggccgcgtgc
301 ggcggtttct gggggagggg gctcgggtga ttcagcgccc ggtcaggcgg aagcggccgc
361 cgccgcggag gaggaggaga aagaggaggg gacagctgtc cgcgcccggg cgcccgcggt
421 ggcacgagcc cgcggcccga gtgcgaggcg gaggcgagga ggccgcgggg acgcgaggcg
481 aggccggccg gggcccccgc agccatggag aacgctcaca caaagaccgt ggaggaggtg
541 ctgggccact tcggcgtcaa cgagagcacg gggctgagcc tggagcaggt caagaagctc
601 aaggagagat ggggctccaa cgaattgccg gctgaagaag gaaagacctt gctggaactt
661 gtgatcgagc agtttgaaga cttactagtt agaattttat tgctggcagc atgtatatct
721 ttcgttttgg cttggttcga agaaggtgaa gaaacgatta cagcctttgt agaacctttt
781 gtaattctgc ttatattggt agccaatgca attgtgggtg tatggcagga gagaaacgct
841 gaaaatgcaa tagaagctct taaggagtat gaacctgaaa tgggcaaggt gtatcgacag
901 gacagaaaga gtgtgcagcg gattaaagcg aaagatatag ttcctgggga tatagtggaa
961 attgctgttg gtgacaaagt tccggctgac attagattga catccatcaa gtctacaact
1021 ctgagagttg accagtcgat tcttacaggt gaatctgtct cggtcatcaa gcatactgac
1081 cccgtccctg acccacgagc tgttaatcaa gacaaaaaga acatgctctt ttctggcaca
1141 aacatcgctg ctggcaaagc tatgggagtg gtggtggcga ctggagtcaa tactgagatc
1201 ggcaagatcc gggatgaaat ggttgcaaca gaacaggaga gaacacccct acagcagaag
1261 ctggacgagt ttggggaaca gctttccaaa gttatctccc tcatttgcat tgcagtctgg
1321 atcatcaaca tcgggcattt caatgaccca gttcatggtg gctcttggat cagaggtgcc
1381 atctactact ttaagattgc agtggccctg gctgttgctg ccatccctga gggtctgcct
1441 gctgtcatca ccacctgctt ggctcttgga actcgaagga tggcaaagaa aaatgctatt
1501 gttcgaagtc tgccttctgt ggaaaccctt ggttgtactt ctgttatctg ctcagacaag
1561 accggcacac ttaccacaaa ccagatgtcc gtctgcagga tgttcattct ggacaaagta
1621 gaaggtgata cttgttccct taatgagttt accataactg gatcaaccta tgcacccatt
1681 ggagaagtgc aaaaagatga caagccagtg aaatgccatc agtatgacgg gcttgtagag
1741 ttagcaacga tctgtgctct gtgtaatgac tctgctttgg attacaatga ggcgaagggt
1801 gtgtatgaaa aagttggaga agccacagag actgctctca cgtgcctggt agagaagatg
1861 aatgtattcg acacggagct gaagggactt tctaaaatag aacgcgccaa cgcctgcaac
1921 tcggtcataa aacagcttat gaagaaggaa ttcacgctag agttttcacg tgatagaaaa
1981 tcaatgtccg tctactgtac accaaacaaa ccgagccgga cgtccatgag caagatgttt
2041 gtgaagggtg ctccagaagg tgtcatcgac aggtgcaccc acatccgagt tggaagtacc
2101 aaggtcccca tgacgcctgg tgttaaacag aagattatgt ctgtcattcg ggagtggggc
2161 agtggcagcg acacactgcg gtgcctggct ctggccactc atgacaaccc gctgaggaga
2221 gaggagatgc acctggaaga ttctgcgaac ttcatcaaat atgagaccaa tctgactttc
2281 gttggctgtg tgggcatgct ggaccctccc aggattgaag tggcctcttc tgtgaagctg
2341 tgccggcaag cgggcatccg agtcatcatg atcactgggg ataacaaagg cactgctgtg
2401 gccatctgtc gccgcattgg catctttggg caggatgagg atgtgacatc aaaggctttt
2461 acagggcgag aatttgatga attaagcccc tcagcccaga gagacgcctg cttaaatgcc
2521 cgttgttttg ctcgagttga accttcccac aagtctaaga tcgttgagtt cctgcagtcc
2581 tttgatgaga tcacagctat gactggtgat ggtgtgaacg acgcgcccgc tctgaagaag
2641 tcggaaatcg ggattgccat gggctcaggg actgcagtgg ctaagacggc ctctgagatg
2701 gtgctggccg acgacaactt ctccaccatc gtggccgctg ttgaggaggg gcgcgccatc
2761 tacaacaaca tgaagcagtt catccgctac ctcatctcct ccaacgtggg ggaggtggtc
2821 tgtatcttcc tgacggcagc ccttgggttt cctgaggctt tgattcctgt ccagttactc
2881 tgggtcaatc tggtgacgga tggtctgcct gccactgcgc tggggttcaa tcctccagat
2941 ctggacatca tgaacaaacc cccacggaac ccaaaagaac cgctgatcag cgggtggctc
3001 tttttccgtt acctggctat tggctgttat gttggcgctg ccaccgtggg tgctgctgcg
3061 tggtggttca tcgctgctga cggtggtccg agagtctcct tctaccagct gagtcatttc
3121 ctgcagtgta aggaggacaa cccagacttc gaaggagtgg attgtgcaat ctttgagtcc
3181 ccgtatccga tgacaatggc actttctgtt ctagtcacca tagagatgtg caatgccctc
3241 aacagcttgt ctgaaaacca gtccctgctg aggatgcccc cctgggagaa catctggctc
3301 gtgggctcca tctgcttgtc catgtccctt cacttcttga tcctctacgt ggaacctttg
3361 ccactcattt tccagatcac accgctgaat ctgacccagt ggctgatggt gctgaaaatc
3421 tccctgcctg tgatcctcat ggacgagacg ctcaagtttg tggcccgaaa ctacctggag
3481 cctggtaaag agtgtgcgca gcctgccacc aaaccctcct gctccctgtc ggcatgcacc
3541 gacggcattt cctggccctt tgtgctgctc attatgcccc tggtggtctg ggtctacagc
3601 acagacacta actttagtga tatgttctgg tcttgactga cagcgctaca tgcagaagat
3661 gtttaactta atcaattaat tttttattgt ttaaagcaac tgtctatttc tgctgaattt
3721 tcacatgaac atattggctg gtgaaggagg tttcatatct agattttgtt ttgctttttc
3781 tgactccagt gggacaagat tttccttttt tttatacaca taattaaagt gtccattgac
3841 atgtacagag aactaacact attttatgca gatatttttt tgtagatgaa aaaagcatgt
3901 acagtgttct gtttcatact catacttgta caaagatcgt tgagccagca gacattgtca
3961 gcaagttaat tggcagcaga cttaggaaat gaatgtgtgt ggtttttaaa agaaaaaaaa
4021 ctaaatagca tgtattgtgt cttttgcatg attctctgga tttaatttgg tatcacagtc
4081 taatttttat tcataagcca atttttctgc actgagcaga gtcctgctac ctcagtcagt
4141 atttttggtt tgccactccc tgcaccctca gcctctgtca cccccagccc accctggccc
4201 gctcggctgc ttctgtaggt ctgatggttc tgtttacatc tgtcttcacg aggtttgcct
4261 gtctcgcttg tgcagaaaac attgctccag atccaatcga ctgggtttct gtcccttcgc
4321 ctagttttta aggttattta tttaaatgtc taatgtattt tattgtaaca gacattgttt
4381 tgccaacatt gcctattcag tggcacttca caatctagtt taaaagaaaa ataaaacatt
4441 ttaaatggac agaggaaaat aactgtcttg ttcttaatgg ccttgaacta cccatgatca
4501 gttcaaattc ctgagcttct cagctcggag ccttagtttc ctgttcctga gaagaacgtg
4561 tactgctgca cagtagggat tatctataat gtacaggcac gatcgtcatg tgacaaacat
4621 ttactcaagt ttctgctggc ctggtgtatg taggctggtg taagggcaag tgtgtgtgtg
4681 tatgtgtgtg tgtgttctgt aaaatctgta aatagcacat gaccaaatga actgtataga
4741 aatattttta tttgaatatg gcactaacca ccaccaccat gactgaccgg acgtttgcgc
4801 atgttcaagg tcagtcttaa cggcagtgtg agtcattaac agtcctaact gtggtgtttt
4861 cctccaatgc cttccaacat ccatcaacta accaatactg gagtaaccgc ttcctaaaca
4921 ttgcagaaat gtaagggtgt tcgggtgcgt gcatgtgcgt tgttagcaac acatcttcca
4981 gccctctgca tgactgagct tggggaaaga gaaatagaac agcccccagc tcactgtgtg
5041 atgtggagga aatgtgtatt acaagtgggg ttttagctgt tgagtcaaaa taataacaag
5101 tgtacaattt agcataagga atcggagagc ctctccagag aagtcggttt ctttgctgca
5161 agaagaatga ggttctgaac ccttatccaa gaacagaagc catcagccaa gtctccacat
5221 ttctctgcaa aatgttgtag cctctataac tgtatgatag tgtaatgcat gccttcagtt
5281 gtaagtggcc agatcgcgct acagtgacat tgaaacctgc tctctaattg gccctgtaca
5341 gtttgcttat ttataaattc atttaaaaca ctacagctgt tgaatggtta caacctaggc
5401 ctccggtcct aacttcagtt gttctcctcg gtgtgcagcc agctgttcca cactgtatta
5461 ttgtaactta tttagtgaag tcagaagcag tagacagatg ttggtgcaat acaagtattg
5521 tgtgcattta tcgtaataaa gtgctccgcg tcggttcagt tcctcacagc ttctcacagt
5581 gcatgtctga ctgtagtctg taaatagagg tcagtgtccg tgctgctaac aggtatcgat
5641 cgcacaga
