请各位高手帮我看看我的这个片段怎么总是扩增不出来
本人有构建一片段扩增后连接到pcDNA3.1质粒上。先扩增一含有内含子序列的片段，具体如下：
目的： 我现在要把SASH1基因intron12 的5’端的一部分序列（至少50bp）、extron 13的全部序列、intron13
的全部序列、extron14的全部序列和intron14的一部分序列（至少50bp）扩增出来。
引物合成要求：在前向引物的5’端加上保护碱基和HindIII的酶缺序列AAGCTT;在后向引物的5’端加入保护碱基和EcoRV的酶缺序列GATATC，引物的GC％最好在40％-60％之间。
各内含子和外显子序列：
Intron12
gtatgc agctacctgg tagaatgttc ccttggttct tcctgacaag 148890471
aaaagctgag aatggccctt ctgagatttt aaatggtgct aaatacacat 148890521
actccttggc attaacctgc tccaagtccg tgctttattt acgtagagaa 148890571
acactgaatt caacacacgt ttattaagca ctttgggctg ctcagcaccg 148890621
tgcttggtgc tgggaataca aaatacagaa cttgccttct gtgtacagac 148890671
aagagagaca gacatgtaaa gaaatggtta ttcttcagtg cactaagcgt 148890721
tgtaaatttg aatacaaagg aaggatttta ttttctaaac tatgccttgc 148890771
tacaaaatgg atgttacctg aatcaaacct aaagcttttt ttttttgggg 148890821
gggggggtgg cagtagactt gtcgatcata gcataagttg ttctagaagc 148890871
acatagccag tctaagaaat aaactcaaag ttcttcctgg gtaccaggct 148890921
ttggtaataa tgtttcagag aggggtgttt tccctcattt gccttatctg 148890971
tagccatgaa ggaaggtatc atagaattga tttttccccc ctggatctat 148891021
aatttcattg ctagagaaat gactgtttct gtctaaattg catgggtgga 148891071
gggagtaaag atagttggag agctgggcct caaggtaatt atgaccattc 148891121
aattttcaag acactctcct ccagcacctg tatagcactc attttgtagc 148891171
ctgttgaagt tcctgtgtat gagctggttg tctgcttgaa atatgtaggt 148891221
aatgcacttt atctaccaaa taaaaacagc aggaaaagca gacatttttg 148891271
ctcagctgct gctctctggt gggcacagtg agctaaaacc aagcatcgcc 148891321
cctatgcgaa tgaccgaacg tcaccagcct gccacccacc tcacttgatt 148891371
caaacaaagc ctgcacagca gcttcagttg attacagtgg ttctcacggg 148891421
gaaggggtga gaggctatca ttttgctccc ccatggactc cagggcaatg 148891471
tctttcaatg gtcagactgg gttgaggtga agtggagtgg atattgctgg 148891521
catctagggc agcagtgccc aatctttttg caccagggac tggtttcgtg 148891571
gaaggcaatt tttccacgga gctggtgtag gtggtggttt caggatgatt 148891621
cacatgcatt acatttattg ttcacttcat ttctattgtt attacattgt 148891671
aatatataat gaaataatta tacaactcac cataatgtag aatcagtggg 148891721
agccctgagc ttgttttcct gcaactagat gatcccatct ggggatgatg 148891771
ggagacagtg acagatcatc aggcattaga ttctcataaa cagcgtgcaa 148891821
cctagatgcc tcgtgtgcac aattcacaat aggattcgtg ctcctacgag 148891871
aatctaatac cgccgctgat ctgacaggag gtggagctca gacagtaatg 148891921
cgggtaatgg agagtggctg tgaaaacaga tgaagcttgg cctgctcacc 148891971
cgccactcac ctcctgctgt acggcccact tcttaacagg ccacagacgg 148892021
gttctggtcc gtggcccggg agttggagaa tgctgatcta gaggatagag 148892071
gccagggacg ctgccaaaca tcttagcatg cacaggacgg tctgcacagc 148892121
aaagagctgt ccatcaaaat gatcagtgct caggctgaga aaccgtggct 148892171
tgttagacct gcaggagata gatgtctgga ctgccttcag catgtcttat 148892221
ccatgatttt ttttctttat tcttttcatt ctcttgtcct tgtgggtgac 148892271
ggagtgatag tgattctata tttcttccca gcaatgaagc agtcagcatg 148892321
ctagtgagag catgctacta attgaaagca ttttattaag tttgcttttc 148892371
cccaagatag attgaaacag ccactaaaac tcttaagctt tacgttcaca 148892421
gaggttttct gctttatgaa tcagaagcat agaattttaa tgtcattttg 148892471
gcatttttca ataattttac taccatatag caatcttatg tgcttagttc 148892521
tgttttttta aagcagccat ttcacataag ttaaggaata aaagaaacaa 148892571
ctgatcatta taaattttct gtaaagcaaa aataatggaa ggttttatgt 148892621
ggttgttttt tttttgtttt tgtttttgtt ttgagacgga gtgtcactct 148892671
gtcacccagg ctggagtgca atggtgtggc ctcggcttac tgcaacctct 148892721
gcctcctggg ttcaagcgat tctcccacct cagcctcttg agtagctggg 148892771
actacaggca cgtgccacca tacccggcta attattgtat ttttagtaga 148892821
gatggggttt cactacgttg gccaggctgg tctcgaactc ctgacctcgt 148892871
gagccaccca cctcagcctc ccagagtgct gggattacag gcatgagcca 148892921
ctgcgcccac cttgtgtgtt tttttaaaaa aactttttag taaatatacc 148892971
aaccattatg gtagggatat ttgttttctc cgttgctgct atatgcccaa 148893021
tacctggatt agtgcctgtt attgtaggta attcataaac atgtgatgaa 148893071
taaattaata attttttaaa gcatttgaaa atatatcaga gacccacatt 148893121
tactattgct gtttttcaaa ctgcgtgctt ttttcagaag cattacagaa 148893171
gattttaatg ggaaaaaagt tacttatttt atgacgcaga aaggaggaat 148893221
gaattttttt tttttaagga attcatactt tggagcttca gtaacttcct 148893271
aataatgtca gctctagttt tctttgttgt ttttgaggtc gcttgctcac 148893321
tttcctcaca ttgaaagcac taatgtgtgt tggatcagaa gctgctgttg 148893371
ctgatgtcct cagatgtttg caggacctgc ggagctgtgt taaggccctt 148893421
ctgatgactc ggcagcatgc aaatagcagg tttcatttta gctggttttg 148893471
agcccgtgtg gctgcggcca tatgtaatat gccagagact tttctatttt 148893521
ggcaagtaaa caattctcac tgcaaacttt ctgaagtgtg atatcgtcta 148893571
ttgatgtagc accacgaatc aaataagcat gctatactca aatgtgcaaa 148893621
tgaggagccg ctgagaggat aatacttagc tactcaaaat gcaggaagta 148893671
ctgctgagtc actcccatca ggattctggt gtgaaagaga tttcccagct 148893721
ttttactgac aacactttag aaaagtgaaa gaagctaaaa ctttagagta 148893771
acatatggaa attttatggt agactggaaa atagtatgtt tgctgtatat 148893821
gtgggtttta aattcatgtt tccttatcca atgccaataa tttgtacaaa 148893871
tagaaatata agaatctagc caattttttt tttccaggaa tacacttttt 148893921
taaaagtgca tgtttcctat ccaagtagat taaatatcct ctgactacac 148893971
ttccaggtat tctagtaatc atggattgta cttgatctac atttgaagtc 148894021
cagccttgct actgggcttt tgcgtatgct gagtctctaa acctttaagt 148894071
attttgtctg tgtggtagac agattttttt tttcttggag attccaagaa 148894121
tatctattcg agaatgagaa atagtatttg agtgcttaat aaatagtgac 148894171
attggccacg actttaaaag taagcgagga tgtaagttta agaggaggag 148894221
aatattggtt atagaatcaa atataggtat agattgattt gattgatttc 148894271
gttgaaggcc aagtcgctga gaattacaaa acaacacctg tccactctga 148894321
tgaacttctt cattttgttg aaacccatgg cag―――3929bp
Extron13 （浅蓝色为剪接位点）
GACTCGG GCCTTGATGG 148894371
AATGCCTGGC TCCCCTCCGC CTTCACAGCC CGACCCCGAA CACTTGGACA 148894421
AGCCCAAGCT CAAGGCCGGG GGTTCTGTAG AAAGTCTTCG CAGTTCTCTC 148894471
AGTGGGCAGA GCTCCATGA―――136bp
intron13
g taagtcgagt ttgtcattgt agattatttt 148894521
ctttggagtt aatatctgac atatactgag cactaggttc aggcaatttc 148894571
aaggtgaata agaccaagtc ctgcccttca ggtgctatta gagaatcaga 148894621
ccaataaact ctctggactc agagtcgtgt ttgtttatta gtagacatgc 148894671
agcagcagca gcccaaaaca ggaggtgatg aattttgatc ggagacaata 148894721
gggaaagcat ctcagaggaa ggatctagag agcaggggga ataagccagg 148894771
gagacaagca catgaagatg accagcaaaa taaagggagt ggggaaggga 148894821
ggggaatttt atatgtgact gataaattcg ggccaatggt agttggagtt 148894871
ggctgaatgg tatgaaggaa aagatcaact ttattttatt tttattttta 148894921
tttttgagat ggagtctcac tgtctcccag gctggagtgc agtgttgtga 148894971
tctcagctca ctgcaacctt cacctccctg gctcaaggga tcctcctgcc 148895021
tcagccttcc gcatagctgt actacagggt catgccacca cacccggcta 148895071
attttgttgt agagacgggg ttttgccatg tagcccaggc tggtggaact 148895121
cctggactca agcgatctgc ccacctcggc ctcctaaagt gctgggatta 148895171
taggcgtgag ccaccgtgcc ccgagatcaa gtttaaacct accactttaa 148895221
gctctttctt acccttttgg gcaaatgaca tgactgctct gaatttgttt 148895271
cctcatctgt aaaatggtta gagagagtgt gcattaccac tgagaggtca 148895321
cgaatgttaa gagcagagtc tggtgcctgg ccctgcagtg tcctccacgc 148895371
gggatcctcc gtgccacatg gattcccagg tcatgagggt aacttgctgt 148895421
gagtcccctg tccactgagg agctgaggga tagcactcgg agaattcata 148895471
actgggccct gccctggtcc tgacagaggg ttgtggctca aggcttcctt 148895521
tctctgggcc ttcatttcct aatctgccat accttcaata cctttctgct 148895571
ttgctgggtg ggaaatctgg atctacccgt gttcttccta tgtttcctgt 148895621
acag―――1135bp
extron14（浅蓝色为剪接位点）
GCGGTC AAACAGTGAG CACCACTGAT TCCTCAACCA GCAACCGGGA 148895671
AAGCGTCAAG TCGGAAGATG GGGATGACGA AGAGCCGCCT TACCGAGGCC 148895721
CGTTCTGCGG GCGTGCCAGG GTGCACACCG ACTTCACCCC CAGTCCCTAT 148895771
GACACAGACT CACTCAAGCT CAAG―――170bp
Intron14
gtatct ctctccctgg cctcagagca 148895821
gctaactggg ctcttccatt tctctaggag gctttctttc ctcctcactg 148895871
ttgaatgctg ggctactatg gtacagactg gacagtatct tggctacctc 148895921
gatcactggt ctcctctgta gtgaaaaaca gaacaagaac cagccagccc 148895971
tcgaggcaga gggagggtct cgtgttaatc cctgggttag catcacttca 148896021
cccctctgac ctcagcttcc ctctagaatt cctgttgcac accctccctc 148896071
tccccacctg gctcctgctc cacacacctg gtttctgtgt tttctccatc 148896121
tgaagaaaga tttctgcctt ctgtgtgctc agaggtacct ttatgggaca 148896171
gatggggttc cacaaagtgt gtgcagccat gacagggccg ggattgccac 148896221
tggttaggac aggtgggaag tgcctcctct aagaagtggg agccctctga 148896271
ggagggaagg cgttcagagt gccgggcggg cagcctggct gagagcacca 148896321
ggggtacccc aggcacccct ggttctgcag ggcaggaggg tggaggggct 148896371
gtgaccgggg gcctgcgtgg aattccgtac agtcagagac acctgtctgg 148896421
cctctgcgga gctcacagtc acatcctatg caggtcactc agaggggtga 148896471
cttgtgggac cccgattctg gcctttgtgg catcttcact tctgcatgac 148896521
ctgtgtatct gacagattct tgatttgtac gttcatggaa tgtacctaat 148896571
ggaaagatct ttgctccctg ggccacag―――804 bp
我应用过的引物序列：sash1-3 F ATAT AAGCTT TGA AAC CCA TGG CAG GAC TC
SAsh1-3 R ATAT TCTAGA GAG TGA CAA CTT ACG ACC CG
SASH1-1 F 5’- 36 GCGC AAGCTT TGA AAC CCA TGG CAG GAC TC-3’
SASH1-1 R 5’ 1641—CCCGGTCTAGA GAG TGA CAA CTT ACG ACC CG
SASH1-2 F 5’ 140- GCGC AAGCTT CTT GAT GGA ATG CCT GGC TC –159 3
我要扩增的目的片段：1 TGAAACCCAT GGCAGGACTC GGGCCTTGAT GGAATGCCTG GCTCCCCTCC GCCTTCACAG
61 CCCGACCCCG AACACTTGGA CAAGCCCAAG CTCAAGGCCG GGGGTTCTGT AGAAAGTCTT
121 CGCAGTTCTC TCAGTGGGCA GAGCTCCATG AGTAAGTCGA GTTTGTCATT GTAGATTATT
181 TTCTTTGGAG TTAATATCTG ACATATACTG AGCACTAGGT TCAGGCAATT TCAAGGTGAA
241 TAAGACCAAG TCCTGCCCTT CAGGTGCTAT TAGAGAATCA GACCAATAAA CTCTCTGGAC
301 TCAGAGTCGT GTTTGTTTAT TAGTAGACAT GCAGCAGCAG CAGCCCAAAA CAGGAGGTGA
361 TGAATTTTGA TCGGAGACAA TAGGGAAAGC ATCTCAGAGG AAGGATCTAG AGAGCAGGGG
421 GAATAAGCCA GGGAGACAAG CACATGAAGA TGACCAGCAA AATAAAGGGA GTGGGGAAGG
481 GAGGGGAATT TTATATGTGA CTGATAAATT CGGGCCAATG GTAGTTGGAG TTGGCTGAAT
541 GGTATGAAGG AAAAGATCAA CTTTATTTTA TTTTTATTTT TATTTTTGAG ATGGAGTCTC
601 ACTGTCTCCC AGGCTGGAGT GCAGTGTTGT GATCTCAGCT CACTGCAACC TTCACCTCCC
661 TGGCTCAAGG GATCCTCCTG CCTCAGCCTT CCGCATAGCT GTACTACAGG GTCATGCCAC
721 CACACCCGGC TAATTTTGTT GTAGAGACGG GGTTTTGCCA TGTAGCCCAG GCTGGTGGAA
781 CTCCTGGACT CAAGCGATCT GCCCACCTCG GCCTCCTAAA GTGCTGGGAT TATAGGCGTG
841 AGCCACCGTG CCCCGAGATC AAGTTTAAAC CTACCACTTT AAGCTCTTTC TTACCCTTTT
901 GGGCAAATGA CATGACTGCT CTGAATTTGT TTCCTCATCT GTAAAATGGT TAGAGAGAGT
961 GTGCATTACC ACTGAGAGGT CACGAATGTT AAGAGCAGAG TCTGGTGCCT GGCCCTGCAG
1021 TGTCCTCCAC GCGGGATCCT CCGTGCCACA TGGATTCCCA GGTCATGAGG GTAACTTGCT
1081 GTGAGTCCCC TGTCCACTGA GGAGCTGAGG GATAGCACTC GGAGAATTCA TAACTGGGCC
1141 CTGCCCTGGT CCTGACAGAG GGTTGTGGCT CAAGGCTTCC TTTCTCTGGG CCTTCATTTC
1201 CTAATCTGCC ATACCTTCAA TACCTTTCTG CTTTGCTGGG TGGGAAATCT GGATCTACCC
1261 GTGTTCTTCC TATGTTTCCT GTACAGGCGG TCAAACAGTG AGCACCACTG ATTCCTCAAC
1321 CAGCAACCGG GAAAGCGTCA AGTCGGAAGA TGGGGATGAC GAAGAGCCGC CTTACCGAGG
1381 CCCGTTCTGC GGGCGTGCCA GGGTGCACAC CGACTTCACC CCCAGTCCCT ATGACACAGA
1441 CTCACTCAAG CTCAAGGTAT CTCTCTCCCT GGCCTCAGAG CAGCTAACTG GGCTCTTCCA
1501 TTTCTCTAGG AGGCTTTCTT TCCTCCTCAC TGTTGAATGC TGGGC
本人先用了天根的2×pfu PCR mastermix，后用takara的primestar HS DNA polymerase退火温度做几次了调整，同时有改用touch down PCR还是没有作出来。我做了几次都没有作出来，曾将上面拟扩增的目的片段发给上海捷瑞公司引物合成部工作人员请他们设计引物，但他们说设计不出来。各个大侠，我分析了一下我的引物序列，在没有加酶缺位点和保护碱基之前是没有发夹结构，二聚体之类的，加了酶缺位点和保护碱基后，sash1-3 引物序列只有二聚体存在，但是可以通过预变性排掉干扰。我实在没辙啦，要么重新设计引物，请各位大侠帮帮忙，十万火急！
