PCR仪

我的参数也改了很多,但是还是说"ERROR",请帮忙找一下,谢谢!
1 gccctcgccg cccgcggcgc cccgagcgct ttgtgagcag atgcggagcc gagtggaggg
61 cgcgagccag atgcggggcg acagctgact tgctgagagg aggcggggag gcgcggagcg
121 cgcgtgtggt ccttgcgccg ctgacttctc cactggttcc tgggcaccga aagataaacc
181 tctcataatg aaggcccccg ctgtgcttgc acctggcatc ctcgtgctcc tgtttacctt
241 ggtgcagagg agcaatgggg agtgtaaaga ggcactagca aagtccgaga tgaatgtgaa
301 tatgaagtat cagcttccca acttcaccgc ggaaacaccc atccagaatg tcattctaca
361 tgagcatcac attttccttg gtgccactaa ctacatttat gttttaaatg aggaagacct
421 tcagaaggtt gctgagtaca agactgggcc tgtgctggaa cacccagatt gtttcccatg
481 tcaggactgc agcagcaaag ccaatttatc aggaggtgtt tggaaagata acatcaacat
541 ggctctagtt gtcgacacct actatgatga tcaactcatt agctgtggca gcgtcaacag
601 agggacctgc cagcgacatg tctttcccca caatcatact gctgacatac agtcggaggt
661 tcactgcata ttctccccac agatagaaga gcccagccag tgtcctgact gtgtggtgag
721 cgccctggga gccaaagtcc tttcatctgt aaaggaccgg ttcatcaact tctttgtagg
781 caataccata aattcttctt atttcccaga tcatccattg cattcgatat cagtgagaag
841 gctaaaggaa acgaaagatg gttttatgtt tttgacggac cagtcctaca ttgatgtttt
901 acctgagttc agagattctt accccattaa gtatgtccat gcctttgaaa gcaacaattt
961 tatttacttc ttgacggtcc aaagggaaac tctagatgct cagacttttc acacaagaat
1021 aatcaggttc tgttccataa actctggatt gcattcctac atggaaatgc ctctggagtg
1081 tattctcaca gaaaagagaa aaaagagatc cacaaagaag gaagtgttta atatacttca
1141 ggctgcgtat gtcagcaagc ctggggccca gcttgctaga caaataggag ccagcctgaa
1201 tgatgacatt cttttcgggg tgttcgcaca aagcaagcca gattctgccg aaccaatgga
1261 tcgatctgcc atgtgtgcat tccctatcaa atatgtcaac gacttcttca acaagatcgt
1321 caacaaaaac aatgtgagat gtctccagca tttttacgga cccaatcatg agcactgctt
1381 taataggaca cttctgagaa attcatcagg ctgtgaagcg cgccgtgatg aatatcgaac
1441 agagtttacc acagctttgc agcgcgttga cttattcatg ggtcaattca gcgaagtcct
1501 cttaacatct atatccacct tcattaaagg agacctcacc atagctaatc ttgggacatc
1561 agagggtcgc ttcatgcagg ttgtggtttc tcgatcagga ccatcaaccc ctcatgtgaa
1621 ttttctcctg gactcccatc cagtgtctcc agaagtgatt gtggagcata cattaaacca
1681 aaatggctac acactggtta tcactgggaa gaagatcacg aagatcccat tgaatggctt
1741 gggctgcaga catttccagt cctgcagtca atgcctctct gccccaccct ttgttcagtg
1801 tggctggtgc cacgacaaat gtgtgcgatc ggaggaatgc ctgagcggga catggactca
1861 acagatctgt ctgcctgcaa tctacaaggt tttcccaaat agtgcacccc ttgaaggagg
1921 gacaaggctg accatatgtg gctgggactt tggatttcgg aggaataata aatttgattt
1981 aaagaaaact agagttctcc ttggaaatga gagctgcacc ttgactttaa gtgagagcac
2041 gatgaataca ttgaaatgca cagttggtcc tgccatgaat aagcatttca atatgtccat
2101 aattatttca aatggccacg ggacaacaca atacagtaca ttctcctatg tggatcctgt
2161 aataacaagt atttcgccga aatacggtcc tatggctggt ggcactttac ttactttaac
2221 tggaaattac ctaaacagtg ggaattctag acacatttca attggtggaa aaacatgtac
2281 tttaaaaagt gtgtcaaaca gtattcttga atgttatacc ccagcccaaa ccatttcaac
2341 tgagtttgct gttaaattga aaattgactt agccaaccga gagacaagca tcttcagtta
2401 ccgtgaagat cccattgtct atgaaattca tccaaccaaa tcttttatta gtggtgggag
2461 cacaataaca ggtgttggga aaaacctgaa ttcagttagt gtcccgagaa tggtcataaa
2521 tgtgcatgaa gcaggaagga actttacagt ggcatgtcaa catcgctcta attcagagat
2581 aatctgttgt accactcctt ccctgcaaca gctgaatctg caactccccc tgaaaaccaa
2641 agcctttttc atgttagatg ggatcctttc caaatacttt gatctcattt atgtacataa
2701 tcctgtgttt aagccttttg aaaagccagt gatgatctca atgggcaatg aaaatgtact
2761 ggaaattaag ggaaatgata ttgaccctga agcagttaaa ggtgaagtgt taaaagttgg
2821 aaataagagc tgtgagaata tacacttaca ttctgaagcc gttttatgca cggtccccaa
2881 tgacctgctg aaattgaaca gcgagctaaa tatagagtgg aagcaagcaa tttcttcaac
2941 cgtccttgga aaagtaatag ttcaaccaga tcagaatttc acaggattga ttgctggtgt
3001 tgtctcaata tcaacagcac tgttattact acttgggttt ttcctgtggc tgaaaaagag
3061 aaagcaaatt aaagatctgg gcagtgaatt agttcgctac gatgcaagag tacacactcc
3121 tcatttggat aggcttgtaa gtgcccgaag tgtaagccca actacagaaa tggtttcaaa
3181 tgaatctgta gactaccgag ctacttttcc agaagatcag tttcctaatt catctcagaa
3241 cggttcatgc cgacaagtgc agtatcctct gacagacatg tcccccatcc taactagtgg
3301 ggactctgat atatccagtc cattactgca aaatactgtc cacattgacc tcagtgctct
3361 aaatccagag ctggtccagg cagtgcagca tgtagtgatt gggcccagta gcctgattgt
3421 gcatttcaat gaagtcatag gaagagggca ttttggttgt gtatatcatg ggactttgtt
3481 ggacaatgat ggcaagaaaa ttcactgtgc tgtgaaatcc ttgaacagaa tcactgacat
3541 aggagaagtt tcccaatttc tgaccgaggg aatcatcatg aaagatttta gtcatcccaa
3601 tgtcctctcg ctcctgggaa tctgcctgcg aagtgaaggg tctccgctgg tggtcctacc
3661 atacatgaaa catggagatc ttcgaaattt cattcgaaat gagactcata atccaactgt
3721 aaaagatctt attggctttg gtcttcaagt agccaaaggc atgaaatatc ttgcaagcaa
3781 aaagtttgtc cacagagact tggctgcaag aaactgtatg ctggatgaaa aattcacagt
3841 caaggttgct gattttggtc ttgccagaga catgtatgat aaagaatact atagtgtaca
3901 caacaaaaca ggtgcaaagc tgccagtgaa gtggatggct ttggaaagtc tgcaaactca
3961 aaagtttacc accaagtcag atgtgtggtc ctttggcgtg ctcctctggg agctgatgac
4021 aagaggagcc ccaccttatc ctgacgtaaa cacctttgat ataactgttt acttgttgca
4081 agggagaaga ctcctacaac ccgaatactg cccagacccc ttatatgaag taatgctaaa
4141 atgctggcac cctaaagccg aaatgcgccc atccttttct gaactggtgt cccggatatc
4201 agcgatcttc tctactttca ttggggagca ctatgtccat gtgaacgcta cttatgtgaa
4261 cgtaaaatgt gtcgctccgt atccttctct gttgtcatca gaagataacg ctgatgatga
4321 ggtggacaca cgaccagcct ccttctggga gacatcatag tgctagtact atgtcaaagc
4381 aacagtccac actttgtcca atggtttttt cactgcctga cctttaaaag gccatcgata
4441 ttctttgctc ttgccaaaat tgcactatta taggacttgt attgttattt aaattactgg
4501 attctaagga atttcttatc tgacagagca tcagaaccag aggcttggtc ccacaggcca
4561 cggaccaatg gcctgcagcc gtgacaacac tcctgtcata ttggagtcca aaacttgaat
4621 tctgggttga attttttaaa aatcaggtac cacttgattt catatgggaa attgaagcag
4681 gaaatattga gggcttcttg atcacagaaa actcagaaga gatagtaatg ctcaggacag
4741 gagcggcagc cccagaacag gccactcatt tagaattcta gtgtttcaaa acacttttgt
4801 gtgttgtatg gtcaataaca tttttcatta ctgatggtgt cattcaccca ttaggtaaac
4861 attccctttt aaatgtttgt ttgttttttg agacaggatc tcactctgtt gccagggctg
4921 tagtgcagtg gtgtgatcat agctcactgc aacctccacc tcccaggctc aagcctcccg
4981 aatagctggg actacaggcg cacaccacca tccccggcta atttttgtat tttttgtaga
5041 gacggggttt tgccatgttg ccaaggctgg tttcaaactc ctggactcaa gaaatccacc
5101 cacctcagcc tcccaaagtg ctaggattac aggcatgagc cactgcgccc agcccttata
5161 aatttttgta tagacattcc tttggttgga agaatattta taggcaatac agtcaaagtt
5221 tcaaaatagc atcacacaaa acatgtttat aaatgaacag gatgtaatgt acatagatga
5281 cattaagaaa atttgtatga aataatttag tcatcatgaa atatttagtt gtcatataaa
5341 aacccactgt ttgagaatga tgctactctg atctaatgaa tgtgaacatg tagatgtttt
5401 gtgtgtattt ttttaaatga aaactcaaaa taagacaagt aatttgttga taaatatttt
5461 taaagataac tcagcatgtt tgtaaagcag gatacatttt actaaaaggt tcattggttc
5521 caatcacagc tcataggtag agcaaagaaa gggtggatgg attgaaaaga ttagcctctg
5581 tctcggtggc aggttcccac ctcgcaagca attggaaaca aaacttttgg ggagttttat
5641 tttgcattag ggtgtgtttt atgttaagca aaacatactt tagaaacaaa tgaaaaaggc
5701 aattgaaaat cccagctatt tcacctagat ggaatagcca ccctgagcag aactttgtga
5761 tgcttcattc tgtggaattt tgtgcttgct actgtatagt gcatgtggtg taggttactc
5821 taactggttt tgtcgacgta aacatttaaa gtgttatatt ttttataaaa atgtttattt
5881 ttaatgatat gagaaaaatt ttgttaggcc acaaaaacac tgcactgtga acattttaga
5941 aaaggtatgt cagactggga ttaatgacag catgattttc aatgactgta aattgcgata
6001 aggaaatgta ctgattgcca atacacccca ccctcattac atcatcagga cttgaagcca
6061 agggttaacc cagcaagcta caaagagggt gtgtcacact gaaactcaat agttgagttt
6121 ggctgttgtt gcaggaaaat gattataact aaaagctctc tgatagtgca gagacttacc
6181 agaagacaca aggaattgta ctgaagagct attacaatcc aaatattgcc gtttcataaa
6241 tgtaataagt aatactaatt cacagagtat tgtaaatggt ggatgacaaa agaaaatctg
6301 ctctgtggaa agaaagaact gtctctacca gggtcaagag catgaacgca tcaatagaaa
6361 gaactcgggg aaacatccca tcaacaggac tacacacttg tatatacatt cttgagaaca
6421 ctgcaatgtg aaaatcacgt ttgctattta taaacttgtc cttagattaa tgtgtctgga
6481 cagattgtgg gagtaagtga ttcttctaag aattagatac ttgtcactgc ctatacctgc
6541 agctgaactg aatggtactt cgtatgttaa tagttgttct gataaatcat gcaattaaag
6601 taaagtgatg caacatcttg taaaaaaaaa aaaaaaaaaa a

These primers are for PCR
Forward-Primer ACTGGCATTGCACCCTTCC Reverse-Primer GTTGCCAGAATTCTCTGCACG
Length: 19 Length: 21
Percent GC: 57 Percent GC: 52
Tm: 57 Tm: 57
Self-Anneal: 12 Self-Anneal: 20
End-Anneal: 8 End-Anneal: 8
Offset: 1 Offset: 353

Pair-Anneal: 20
Pair-End-Anneal: 6
Total valid primer pairs: 1
谢谢楼主，请问下设计出来的引物如上，Tm值是否偏低？

我按你的方法，在Select YES to Amplify a region with EXACT endpoints of the DNA sequence entered NO YES这一项中，选择yes则得不到结果，但是选择no每次都能得到结果，选择no得到的结果有什么问题？

你好
我现在设计SMAD3的引物,用那个软件设计,改了很多参数还是不行,加1则多,减1收少,不知如何是好,请求援助,多谢!
序列: 1 ccgcgtggag ccgctcgggc gccgccgtgt tccggatccc cgcaactgca gtgccgctat
61 cctggccggg acgcctgggc aagttctcca gagttaaaag cgaagttcgg gcgcggcgca
121 ggccgagctg cctctgtgcg cccccggcgt ccccagtgcg tccagccccg ccgggggcgc
181 cggtgaccct tccgtgccag ccatgtcgtc catcctgccc ttcacccccc cgatcgtgaa
241 gcgcctgctg ggctggaaga agggcgagca gaacgggcag gaggagaagt ggtgcgagaa
301 ggcggtcaag agcttggtga agaagctcaa gaagacgggg cagttggacg agctggagaa
361 ggccatcacc acgcagaacg tgaacaccaa gtgcattacc atccccaggt cactggatgg
421 ccggctgcag gtatcccatc ggaaggggct cccccatgtc atctactgcc gcttgtggcg
481 gtggcccgac ctgcacagcc accatgagtt acgggccatg gagctctgtg agttcgcctt
541 caacatgaag aaggatgaag tgtgtgtaaa tccttaccac tatcagagag tagagacacc
601 agtgctacct ccagtgttgg tgccacgcca caccgagatc ccggccgagt tcccccctct
661 ggatgactac agccattcca tccccgagaa cactaacttc cccgctggca ttgaacccca
721 gagcaatatt ccagaaaccc cacctcctgg ctacctgagt gaagatggag aaactagcga
781 ccaccagatg aaccacagca tggacgcagg ctctccaaac ctctccccga atccgatgtc
841 cccagcacac aataacttgg acctacagcc agtcacctac tgtgagccgg ccttttggtg
901 ctccatctcc tactacgagc tgaaccagcg agttggggag acattccacg cttcacagcc
961 gtccatgaca gtagatggct tcactgatcc ctccaattca gagcgcttct gtctgggcct
1021 gctgtccaat gttaaccgga atgcagccgt ggagcttaca aggcggcaca ttgggagggg
1081 tgtgcggctc tactacattg gaggggaggt ctttgcagag tgcctcagtg acagtgctat
1141 tttcgtccag tctcccaact gcaaccagcg ctatggctgg caccctgcca ctgtctgcaa
1201 gataccccca ggctgcaacc tgaagatctt caacaaccag gaatttgctg ccctcctagc
1261 tcagtctgtc aaccagggct ttgaggctgt ctaccagttg actcgcatgt gcaccatccg
1321 catgagcttc gtcaaaggct ggggagccga gtacaggaga cagacagtga ccagcacccc
1381 ctgctggatc gagctacacc tgaatgggcc cttgcagtgg cttgacaagg tcctcaccca
1441 gatgggttcc cccagcatcc gctgttccag cgtgtcttag agacactggg agtaaaggga
1501 tcgggttggg gagggcgggc ttggggaaaa tgaccttgga agataactcc atccaacttg
1561 gtcttgtcaa agaacacaga ttctctcaac taaggcacta acctgtttct gaaaccacag
1621 aagatgccag cgatggactt tatgaacagc tgtgtctgct gcatatatgt gccccaggct
1681 gaaggccgag tgtgatggct tctgatctgg tggcttgagc taacaggtgg tgtgtcgcca
1741 cctgactcct tgtttagtga cagagatatg ggatgtcaca gtccaaaaag aaagtgcctt
1801 tctccaggac tggagtgtgg agttcacctt tggagaagaa cttttaatgg agcatgccct
1861 gtctccccac tctcacagag agggtctgtg tacctaggga actggatggc ctacgcaggt
1921 actttccccc acccccagaa tgcagatggg aacatgctgg agagtaaagc tggtgagaac
1981 cgcttcttcc ttggaaggac ctgctgaagc tcagccatgt atgtgatgtg tagttcctag
2041 tatcacatcg atctcaagag atacggatgt tcaagtgttc gcaacaaggg cagtcgtgga
2101 gtacatgtcc tgggtgaggt ggtaccacat tgaatgaata catctctgga agctggcacc
2161 actttctgat gtgtaggcca tgttggttta tggagacagc tgcattaatg aattcaccta
2221 gcacaggctc tttggatgtc ttctgtttga tgaaaacagt cctgggtaca tatgttggct
2281 ggaaaaccaa agaggaccca gtctctgctt cttgccctga ggtttggaaa ctgagagtta
2341 tagaagacac ttcaccagga ggttaagaca tcccaggctg acatgggcaa atgaaagggc
2401 tattaaagtt tttttacacc ttggaaaatt gcaggcttgg tgcagagacc tgtcatcttc
2461 accctgggat gtaagattac ctggctccgg taaaggattg ccaccaaaaa ctgctgcggt
2521 gatcctgtgt gatgagtact gttcgcagtg gcaatcacag agaaaatgag tataagttat
2581 ttcaactgga aaaagagtcc ctttcctggc tgttccagaa cagcacatag gatcccacct
2641 gcaactggta ctctcttctt ggctgctgtt tctgttgtga aatgaggagc ccatgttttc
2701 gcatggattt cccgtggaag tcctgtcctg ttccagaggg gacaaaagtg caccctccaa
2761 tgtgataaat cttctgatga tccagaggct ctggggcata tcctggtgcc tctcctgcag
2821 gagctgtgcc ctcagaagct ctgggcagga tgtttccagc taggcatgct gatcctcctt
2881 cagacacaga tgcctgtctg agtctctccc aggctgttga tacagaactc aaagacaatt
2941 ctgaacaacc cctcaggttc tctgaagggg cacagacccc gggtgtgaac attgtgccgg
3001 acttgacatg gtctgtggac tgctgcttca gacacaggaa gtcctcggaa ctgcctacct
3061 gttctgcatg ctctgagtca gaacacagag acactttggt gtggcttcta gtattcatgg
3121 tcctctcttc tcagccatgg cagagagaca agtagctcct gtgatgccat tctgaattcc
3181 gagtttaaaa tgagctatga gctgctgcct gattgaaatg tacatcaacc tcaggagatg
3241 tggatggaga tgaccaaaac accaaagaga ggcacaaatg gggcagaggg gacaggaagc
3301 cacctcacct gtcctgagga cttgctgcgc ccaacacctc ctgttgagtt aaatcaacgt
3361 ctcttccaat caacactgtt attctcataa gcacagaccc tggacatcag tgagctcaca
3421 ccgctgtagc agtaaagcca tcaggtgacc acttcttttg caaggacagc agaggctcct
3481 gagaagcaag ctgatcttga gtatctctgc ccaggttctt ggtggcaagg gaaggggaag
3541 agcaacaacc agaaccaagc aagatgcttc gtgacattgg aacctacttc cctcctagta
3601 aagacacaga gcagacctgt ggggagcatg agactggccc tctgctctgt gcgagcactt
3661 cacttcccag atggagcact gaagacagca gcatttaaac gtgggaaaag gaggagagcg
3721 gtcttgcaaa aacatgcaga agggaaatcg ggggatgctg tgctggtgag gggagagaca
3781 ctgcttcagt gacgcactgt ccacagtcct gaagattgct cagtcaagat gtggctggga
3841 aatactagtt agtggcacag aggtcgcagg tggcttccca catttgaatg tcagaatggg
3901 acacaaacaa gagaacgaag tacaaggaaa tgacagcagc agggacacta gtaagtgctg
3961 gagtgaacgt cctcctcctc ttgcgcacac acacagcctg aagattcaac ggaacttggg
4021 aatgagcacc atctttccca ccgtcatgaa ggagagctcc gcactactct aatgaaggct
4081 ctactgtggg tcctacctcc tcaacctgga gtgcgcgcca gaacagtctt ccaacagcag
4141 tgaggactcc aaggaggcgg ctctctgacg tggcctgtaa ccagctttgg caaccacagt
4201 ggaagagcag ggtggtcagc aacagtgcct ccagggtgcc taggagggag ctgcgagcag
4261 tgctgtgtgt gacactcctg aaggccatac ctgaaaggca tgtcgctgct tcctatcccc
4321 actaagtccc tacaatcctc tcccttacca tctactccta ctcactatgc ctaaatatgc
4381 atggaacagc caatgacatt cattcacagg taagggacag tctgagcgct cggggcatat
4441 cttgggttcc tgaatgttgt ttaaaggctg gactccagta cagctttatg ggaattaaat
4501 ggcatccatt tcagcggacg ctgcctctgc caagctccca aagttacaca ggtacaggca
4561 gactgactag aaccctcttt gttggccatt cttcctctca ctaatttgat gctttatttt
4621 cctgtcacat taaatttttt atttaataaa aaaaaaaaaa aaaaaaaaaa aaaaaaa

你好,我的引物设计用你教的软件改了好久,最后改到多一则多,少一则少,只好向你求救了,拜托
序列： 1 ccgcgtggag ccgctcgggc gccgccgtgt tccggatccc cgcaactgca gtgccgctat
61 cctggccggg acgcctgggc aagttctcca gagttaaaag cgaagttcgg gcgcggcgca
121 ggccgagctg cctctgtgcg cccccggcgt ccccagtgcg tccagccccg ccgggggcgc
181 cggtgaccct tccgtgccag ccatgtcgtc catcctgccc ttcacccccc cgatcgtgaa
241 gcgcctgctg ggctggaaga agggcgagca gaacgggcag gaggagaagt ggtgcgagaa
301 ggcggtcaag agcttggtga agaagctcaa gaagacgggg cagttggacg agctggagaa
361 ggccatcacc acgcagaacg tgaacaccaa gtgcattacc atccccaggt cactggatgg
421 ccggctgcag gtatcccatc ggaaggggct cccccatgtc atctactgcc gcttgtggcg
481 gtggcccgac ctgcacagcc accatgagtt acgggccatg gagctctgtg agttcgcctt
541 caacatgaag aaggatgaag tgtgtgtaaa tccttaccac tatcagagag tagagacacc
601 agtgctacct ccagtgttgg tgccacgcca caccgagatc ccggccgagt tcccccctct
661 ggatgactac agccattcca tccccgagaa cactaacttc cccgctggca ttgaacccca
721 gagcaatatt ccagaaaccc cacctcctgg ctacctgagt gaagatggag aaactagcga
781 ccaccagatg aaccacagca tggacgcagg ctctccaaac ctctccccga atccgatgtc
841 cccagcacac aataacttgg acctacagcc agtcacctac tgtgagccgg ccttttggtg
901 ctccatctcc tactacgagc tgaaccagcg agttggggag acattccacg cttcacagcc
961 gtccatgaca gtagatggct tcactgatcc ctccaattca gagcgcttct gtctgggcct
1021 gctgtccaat gttaaccgga atgcagccgt ggagcttaca aggcggcaca ttgggagggg
1081 tgtgcggctc tactacattg gaggggaggt ctttgcagag tgcctcagtg acagtgctat
1141 tttcgtccag tctcccaact gcaaccagcg ctatggctgg caccctgcca ctgtctgcaa
1201 gataccccca ggctgcaacc tgaagatctt caacaaccag gaatttgctg ccctcctagc
1261 tcagtctgtc aaccagggct ttgaggctgt ctaccagttg actcgcatgt gcaccatccg
1321 catgagcttc gtcaaaggct ggggagccga gtacaggaga cagacagtga ccagcacccc
1381 ctgctggatc gagctacacc tgaatgggcc cttgcagtgg cttgacaagg tcctcaccca
1441 gatgggttcc cccagcatcc gctgttccag cgtgtcttag agacactggg agtaaaggga
1501 tcgggttggg gagggcgggc ttggggaaaa tgaccttgga agataactcc atccaacttg
1561 gtcttgtcaa agaacacaga ttctctcaac taaggcacta acctgtttct gaaaccacag
1621 aagatgccag cgatggactt tatgaacagc tgtgtctgct gcatatatgt gccccaggct
1681 gaaggccgag tgtgatggct tctgatctgg tggcttgagc taacaggtgg tgtgtcgcca
1741 cctgactcct tgtttagtga cagagatatg ggatgtcaca gtccaaaaag aaagtgcctt
1801 tctccaggac tggagtgtgg agttcacctt tggagaagaa cttttaatgg agcatgccct
1861 gtctccccac tctcacagag agggtctgtg tacctaggga actggatggc ctacgcaggt
1921 actttccccc acccccagaa tgcagatggg aacatgctgg agagtaaagc tggtgagaac
1981 cgcttcttcc ttggaaggac ctgctgaagc tcagccatgt atgtgatgtg tagttcctag
2041 tatcacatcg atctcaagag atacggatgt tcaagtgttc gcaacaaggg cagtcgtgga
2101 gtacatgtcc tgggtgaggt ggtaccacat tgaatgaata catctctgga agctggcacc
2161 actttctgat gtgtaggcca tgttggttta tggagacagc tgcattaatg aattcaccta
2221 gcacaggctc tttggatgtc ttctgtttga tgaaaacagt cctgggtaca tatgttggct
2281 ggaaaaccaa agaggaccca gtctctgctt cttgccctga ggtttggaaa ctgagagtta
2341 tagaagacac ttcaccagga ggttaagaca tcccaggctg acatgggcaa atgaaagggc
2401 tattaaagtt tttttacacc ttggaaaatt gcaggcttgg tgcagagacc tgtcatcttc
2461 accctgggat gtaagattac ctggctccgg taaaggattg ccaccaaaaa ctgctgcggt
2521 gatcctgtgt gatgagtact gttcgcagtg gcaatcacag agaaaatgag tataagttat
2581 ttcaactgga aaaagagtcc ctttcctggc tgttccagaa cagcacatag gatcccacct
2641 gcaactggta ctctcttctt ggctgctgtt tctgttgtga aatgaggagc ccatgttttc
2701 gcatggattt cccgtggaag tcctgtcctg ttccagaggg gacaaaagtg caccctccaa
2761 tgtgataaat cttctgatga tccagaggct ctggggcata tcctggtgcc tctcctgcag
2821 gagctgtgcc ctcagaagct ctgggcagga tgtttccagc taggcatgct gatcctcctt
2881 cagacacaga tgcctgtctg agtctctccc aggctgttga tacagaactc aaagacaatt
2941 ctgaacaacc cctcaggttc tctgaagggg cacagacccc gggtgtgaac attgtgccgg
3001 acttgacatg gtctgtggac tgctgcttca gacacaggaa gtcctcggaa ctgcctacct
3061 gttctgcatg ctctgagtca gaacacagag acactttggt gtggcttcta gtattcatgg
3121 tcctctcttc tcagccatgg cagagagaca agtagctcct gtgatgccat tctgaattcc
3181 gagtttaaaa tgagctatga gctgctgcct gattgaaatg tacatcaacc tcaggagatg
3241 tggatggaga tgaccaaaac accaaagaga ggcacaaatg gggcagaggg gacaggaagc
3301 cacctcacct gtcctgagga cttgctgcgc ccaacacctc ctgttgagtt aaatcaacgt
3361 ctcttccaat caacactgtt attctcataa gcacagaccc tggacatcag tgagctcaca
3421 ccgctgtagc agtaaagcca tcaggtgacc acttcttttg caaggacagc agaggctcct
3481 gagaagcaag ctgatcttga gtatctctgc ccaggttctt ggtggcaagg gaaggggaag
3541 agcaacaacc agaaccaagc aagatgcttc gtgacattgg aacctacttc cctcctagta
3601 aagacacaga gcagacctgt ggggagcatg agactggccc tctgctctgt gcgagcactt
3661 cacttcccag atggagcact gaagacagca gcatttaaac gtgggaaaag gaggagagcg
3721 gtcttgcaaa aacatgcaga agggaaatcg ggggatgctg tgctggtgag gggagagaca
3781 ctgcttcagt gacgcactgt ccacagtcct gaagattgct cagtcaagat gtggctggga
3841 aatactagtt agtggcacag aggtcgcagg tggcttccca catttgaatg tcagaatggg
3901 acacaaacaa gagaacgaag tacaaggaaa tgacagcagc agggacacta gtaagtgctg
3961 gagtgaacgt cctcctcctc ttgcgcacac acacagcctg aagattcaac ggaacttggg
4021 aatgagcacc atctttccca ccgtcatgaa ggagagctcc gcactactct aatgaaggct
4081 ctactgtggg tcctacctcc tcaacctgga gtgcgcgcca gaacagtctt ccaacagcag
4141 tgaggactcc aaggaggcgg ctctctgacg tggcctgtaa ccagctttgg caaccacagt
4201 ggaagagcag ggtggtcagc aacagtgcct ccagggtgcc taggagggag ctgcgagcag
4261 tgctgtgtgt gacactcctg aaggccatac ctgaaaggca tgtcgctgct tcctatcccc
4321 actaagtccc tacaatcctc tcccttacca tctactccta ctcactatgc ctaaatatgc
4381 atggaacagc caatgacatt cattcacagg taagggacag tctgagcgct cggggcatat
4441 cttgggttcc tgaatgttgt ttaaaggctg gactccagta cagctttatg ggaattaaat
4501 ggcatccatt tcagcggacg ctgcctctgc caagctccca aagttacaca ggtacaggca
4561 gactgactag aaccctcttt gttggccatt cttcctctca ctaatttgat gctttatttt
4621 cctgtcacat taaatttttt atttaataaa aaaaaaaaaa aaaaaaaaaa aaaaaaa
//多谢

我是初学者,自己还没有涉及到引物设计,但以后肯定会遇到这个问题,所以提前看看,谢谢楼主的网址,也谢谢大家的启发