请帮忙看看引物设计是否可行,急啊在线等!
丁香园论坛
678
我合成了好几对引物都因为产物的GC含量极高达74%,都没有成功,我又合成了一对引物,没有加酶切位点,想如果能P 出来就回收做摸板,大家请帮我看看,引物设计可行吗?
上游引物:5-ATGGCGGGCGTCTGCTCTCCACCTG-3
下游引物:5-CCACCTTGAAGTCATTCTCCAGGCGGC-3
如果 不行,请大家帮我设计一下引物,感激不尽!我已经做了快半年了.郁闷啊!
产物序列:
1 tgcagcggtt ctggatggcg ttattcgtac ttttgctgcg cccattcttc cctgttccat
61 cgcggtgcgg gcatggcggg cgtctgctct ccacctggca tccaatgaac atgtctctgt
121 cacgcattgt caaggcggcg cccctgcgcc gcaccacgct ggccatggcg ctgggcgcgc
181 tgggcgccgc cccggcggcg catgccgact ggaacaacca gtccatcgtc aagaccggtg
241 agcgccagca tggcatccat atccagggct ccgacccggg cggcgtacgg accgccagcg
301 gaaccaccat caaggtaagc ggccgtcagg cccagggcat cctgctagaa aatcccgcgg
361 ccgagctgca gttccggaac ggcagtgtca cgtcgtcggg acagttgtcc gacgatggca
421 tccggcgctt tctgggcacc gtcaccgtca aggccggcaa gctggtcgcc gatcacgcca
481 cgctggccaa cgttggcgac acctgggacg acgacggcat cgcgctctat gtggccggcg
541 aacaggccca ggccagcatc gccgacagca ccctgcaggg cgctggcggc gtgcagatcg
601 agcgcggcgc caatgtcacg gtccaacgca gcgccatcgt cgacgggggc ttgcatatcg
661 gcgccctgca gtcattgcag ccggaagacc ttccgcccag ccgggtggtg ctgcgcgaca
721 ccaacgtgac cgccgtgccc gccagcggcg cgcccgcggc ggtgtctgtg ttgggggcca
781 gtgagcttac gctcgacggc gggcacatca ccggcgggcg ggcagcgggg gtggcggcca
841 tgcaaggggc ggtcgtgcat ctgcagcgcg cgacgatacg gcgcggggac gcgcctgccg
901 gcggtgcggt tcccggcggt gcggttcccg gtggtgcggt tcccggcggc ttcggtcccg
961 gcggcttcgg tcccgtcctc gacggctggt atggcgtgga cgtatcgggc tccagcgtgg
1021 agctcgccca gtcgatcgtc gaggcgccgg agctgggcgc cgcaatccgg gtgggccgcg
1081 gcgccagggt gacggtgtcg ggcggcagct tgtccgcacc gcacggcaat gtcatcgaga
1141 ccggcggcgc gcgtcgcttt gcgcctcaag ccgcgcccct gtcgatcacc ttgcaggccg
1201 gcgcgcatgc ccaggggaaa gcgctgctgt accgggtcct gccggagccc gtgaagctga
1261 cgctgaccgg gggcgccgat gcgcagggcg acatcgtcgc gacggagctg ccctccattc
1321 ccggcacgtc gatcgggccg ctcgacgtgg cgctggccag ccaggcccga tggacgggcg
1381 ctacccgcgc ggtcgactcg ctgtccatcg acaacgccac ctgggtcatg acggacaact
1441 cgaacgtcgg tgcgctacgg ctggccagcg acggcagcgt cgatttccag cagccggccg
1501 aagctgggcg gttcaaggtc ctgacggtca atacgctggc gggttcgggg ctgttccgca
1561 tgaatgtctt cgcggacctg gggctgagcg acaagctggt cgtcatgcag gacgccagcg
1621 gccagcacag gctgtgggtc cgcaacagcg gcagcgagcc ggccagcgcc aacaccctgc
1681 tgctggtgca gacgccacta ggcagcgcgg cgacctttac ccttgccaac aaggacggca
1741 aggtcgatat cggtacctat cgctatcgat tggccgccaa cggcaatggg cagtggagcc
1801 tggtgggcgc gaaggcgccg ccggcgccca agcccgcgcc gcagccgggt ccccagccgc
1861 cgcagccgcc gcagccgcag ccggaagcgc cggcgccgca accgccggcg ggcagggagt
1921 tgtccgccgc cgccaacgcg gcggtcaaca cgggtggggt gggcctggcc agcacgctct
1981 ggtacgccga aagcaatgcg ttgtccaagc gcctgggcga gttgcgcctg aatccggacg
2041 ccggcggcgc ctggggccgc ggcttcgcgc aacgccagca gctggacaac cgcgccgggc
2101 ggcgcttcga ccagaaggtg gccggcttcg agctgggcgc cgaccacgcg gtggcggtgg
2161 ccggcggacg ctggcacctg ggcgggctgg ccggctatac gcgcggcgac cgcggcttca
2221 ccggcgacgg cggcggccac accgacagcg tgcatgtcgg gggctatgcc acatatatcg
2281 ccgacagcgg tttctacctg gacgcgacgc tgcgcgccag ccgcctggag aatgacttca
2341 aggtggcggg cagcgacggg tacgcggtca agggcaagta ccgcacccat ggggtgggcg
2401 cctcgctcga ggcgggccgg cgctttaccc atgccgacgg ctggttcctc gagccgcagg
2461 ccgagctggc ggtattccgg gccggcggcg gtgcgtaccg cgcggccaac ggcctgcggg
2521 tgcgcgacga aggcggcagc tcggtgctgg gtcgcctggg cctggaggtc ggcaagcgca
2581 tcgaactggc aggcggcagg caggtgcagc catacatcaa ggccagcgtg ctgcaggagt
2641 tcgacggcgc gggtacggta cacaccaacg gcatcgcgca ccgcaccgaa ctgcgcggca
2701 cgcgcgccga actgggcctg ggcatggccg ccgcgctggg ccgcggccac agcctgtatg
2761 cctcgtacga gtactccaag ggcccgaagc tggccatgcc gtggaccttc cacgcgggct
2821 accggtacag ctggtaaagc gaggagggtc tatcccccgc ggaggaggtt ttcctggagc
2881 ttggccggtg ccagtctcca ggctcaggcg gccagggcct gcgggccggg caggccgcgc
2941 tggtgctgg
上游引物:5-ATGGCGGGCGTCTGCTCTCCACCTG-3
下游引物:5-CCACCTTGAAGTCATTCTCCAGGCGGC-3
如果 不行,请大家帮我设计一下引物,感激不尽!我已经做了快半年了.郁闷啊!
产物序列:
1 tgcagcggtt ctggatggcg ttattcgtac ttttgctgcg cccattcttc cctgttccat
61 cgcggtgcgg gcatggcggg cgtctgctct ccacctggca tccaatgaac atgtctctgt
121 cacgcattgt caaggcggcg cccctgcgcc gcaccacgct ggccatggcg ctgggcgcgc
181 tgggcgccgc cccggcggcg catgccgact ggaacaacca gtccatcgtc aagaccggtg
241 agcgccagca tggcatccat atccagggct ccgacccggg cggcgtacgg accgccagcg
301 gaaccaccat caaggtaagc ggccgtcagg cccagggcat cctgctagaa aatcccgcgg
361 ccgagctgca gttccggaac ggcagtgtca cgtcgtcggg acagttgtcc gacgatggca
421 tccggcgctt tctgggcacc gtcaccgtca aggccggcaa gctggtcgcc gatcacgcca
481 cgctggccaa cgttggcgac acctgggacg acgacggcat cgcgctctat gtggccggcg
541 aacaggccca ggccagcatc gccgacagca ccctgcaggg cgctggcggc gtgcagatcg
601 agcgcggcgc caatgtcacg gtccaacgca gcgccatcgt cgacgggggc ttgcatatcg
661 gcgccctgca gtcattgcag ccggaagacc ttccgcccag ccgggtggtg ctgcgcgaca
721 ccaacgtgac cgccgtgccc gccagcggcg cgcccgcggc ggtgtctgtg ttgggggcca
781 gtgagcttac gctcgacggc gggcacatca ccggcgggcg ggcagcgggg gtggcggcca
841 tgcaaggggc ggtcgtgcat ctgcagcgcg cgacgatacg gcgcggggac gcgcctgccg
901 gcggtgcggt tcccggcggt gcggttcccg gtggtgcggt tcccggcggc ttcggtcccg
961 gcggcttcgg tcccgtcctc gacggctggt atggcgtgga cgtatcgggc tccagcgtgg
1021 agctcgccca gtcgatcgtc gaggcgccgg agctgggcgc cgcaatccgg gtgggccgcg
1081 gcgccagggt gacggtgtcg ggcggcagct tgtccgcacc gcacggcaat gtcatcgaga
1141 ccggcggcgc gcgtcgcttt gcgcctcaag ccgcgcccct gtcgatcacc ttgcaggccg
1201 gcgcgcatgc ccaggggaaa gcgctgctgt accgggtcct gccggagccc gtgaagctga
1261 cgctgaccgg gggcgccgat gcgcagggcg acatcgtcgc gacggagctg ccctccattc
1321 ccggcacgtc gatcgggccg ctcgacgtgg cgctggccag ccaggcccga tggacgggcg
1381 ctacccgcgc ggtcgactcg ctgtccatcg acaacgccac ctgggtcatg acggacaact
1441 cgaacgtcgg tgcgctacgg ctggccagcg acggcagcgt cgatttccag cagccggccg
1501 aagctgggcg gttcaaggtc ctgacggtca atacgctggc gggttcgggg ctgttccgca
1561 tgaatgtctt cgcggacctg gggctgagcg acaagctggt cgtcatgcag gacgccagcg
1621 gccagcacag gctgtgggtc cgcaacagcg gcagcgagcc ggccagcgcc aacaccctgc
1681 tgctggtgca gacgccacta ggcagcgcgg cgacctttac ccttgccaac aaggacggca
1741 aggtcgatat cggtacctat cgctatcgat tggccgccaa cggcaatggg cagtggagcc
1801 tggtgggcgc gaaggcgccg ccggcgccca agcccgcgcc gcagccgggt ccccagccgc
1861 cgcagccgcc gcagccgcag ccggaagcgc cggcgccgca accgccggcg ggcagggagt
1921 tgtccgccgc cgccaacgcg gcggtcaaca cgggtggggt gggcctggcc agcacgctct
1981 ggtacgccga aagcaatgcg ttgtccaagc gcctgggcga gttgcgcctg aatccggacg
2041 ccggcggcgc ctggggccgc ggcttcgcgc aacgccagca gctggacaac cgcgccgggc
2101 ggcgcttcga ccagaaggtg gccggcttcg agctgggcgc cgaccacgcg gtggcggtgg
2161 ccggcggacg ctggcacctg ggcgggctgg ccggctatac gcgcggcgac cgcggcttca
2221 ccggcgacgg cggcggccac accgacagcg tgcatgtcgg gggctatgcc acatatatcg
2281 ccgacagcgg tttctacctg gacgcgacgc tgcgcgccag ccgcctggag aatgacttca
2341 aggtggcggg cagcgacggg tacgcggtca agggcaagta ccgcacccat ggggtgggcg
2401 cctcgctcga ggcgggccgg cgctttaccc atgccgacgg ctggttcctc gagccgcagg
2461 ccgagctggc ggtattccgg gccggcggcg gtgcgtaccg cgcggccaac ggcctgcggg
2521 tgcgcgacga aggcggcagc tcggtgctgg gtcgcctggg cctggaggtc ggcaagcgca
2581 tcgaactggc aggcggcagg caggtgcagc catacatcaa ggccagcgtg ctgcaggagt
2641 tcgacggcgc gggtacggta cacaccaacg gcatcgcgca ccgcaccgaa ctgcgcggca
2701 cgcgcgccga actgggcctg ggcatggccg ccgcgctggg ccgcggccac agcctgtatg
2761 cctcgtacga gtactccaag ggcccgaagc tggccatgcc gtggaccttc cacgcgggct
2821 accggtacag ctggtaaagc gaggagggtc tatcccccgc ggaggaggtt ttcctggagc
2881 ttggccggtg ccagtctcca ggctcaggcg gccagggcct gcgggccggg caggccgcgc
2941 tggtgctgg
