GC含量高的序列怎么拉出来
丁香园论坛
1222
谢谢各位的解答，SORRY，这几天没上来，我的序列如下，请帮忙看一下：
NM_190847. Oryza sativa (jap...[gi:34908777] Links
LOCUS NM_190847 1692 bp mRNA linear PLN 07-OCT-2003
DEFINITION Oryza sativa (japonica cultivar-group) putative pectin esterase
(P0415A04.26), mRNA.
1 atgggtatca tccaccgagg ggacagcaca gtgaactgca cgtggctgaa cgaaaaacag
61 aacgggtacc atctaccgac aggtcgatac attcaaaacg cctacccatg caaacttcta
121 tcgttcacgc tgctctcttt agccaccgtg ctctgcctct gcgcgggcgc cgccttcctc
181 ctactctccc cgacggccac gaacctctgc gcgagctccc ctgacccggc ctcgtgccaa
241 gccatcgtcg ccgacgccgt cctagcgtcc ccgcactccc acccgagccg cccggcgcac
301 gtcctccgcg ccatccttgc cacgtctctc gatcggcatg acgcggccgc ggaagcggtg
361 gcgggcatgc gccggcgcgc cagcgacccg aggcaccgcg cggccctgga ggactgcgtc
421 cagctgatgg ggctcgcgcg cgaccgtctc gccgacgcgg cgggcgcgcc cgacgtcgac
481 gtcgacgtcg acgacgcgag gacgtggctc agcgccgtgc tcaccgacca cgtgacgtgc
541 ctggacggcc tcgacgacgg gccgctgcgc gactccgtgg gggcgcacct ggagccgctc
601 aagtccctcg cgagcgcgtc gctcgccgtg ctcagcgccg ccggccgcgg cgctcgggac
661 gtgctcgcgg aggccgtgga caggttcccg tcgtggctga cggcgagaga ccgcacgctc
721 ctcgacgcgg gcgcgggcgc cgtgcaggcg gacgtggtgg tggccaaaga cgggagcggc
781 aagtacacga cgataaagga ggcggtggac gcggcccccg acggcggcaa gagccggtac
841 gtgatctacg tgaagaaggg ggtgtacaag gagaacctcg aggtggggaa gacgaagcgg
901 gtgctgatga tcgtcggcga cggcatggac cagacggtca tcaccggcag ccgcaacgtg
961 gtcgacggct ccacaacttt caactccgcc acgctagcct tgtcgggcga cgggatcatc
1021 ctccaggacc tcaaggtgga gaacacggcg ggcgccgaga agcagcaggc ggtggccctc
1081 cgcgtgagcg ccgaccgcgc cgtgatcaac cgctgccgcc tggacggcta ccaggacacc
1141 ctgtacgcgc accagctccg ccagttctac cgcgactgcg ccgtctccgg cacggtggac
1201 ttcgtgttcg gcaacgccgc ggcggtgctc cagggctgcg tgctcacggc gcggcgcccc
1261 gcgcaggcgc agaagaacgc ggtcacggcg cagggccgga ccgacccgaa ccagaacacc
1321 gggacgtcca tccaccggtg ccgcgtcgtc ccggcgcccg acctggcgcc cgccgcgaag
1381 cagttcccga cgttcctggg ccggccgtgg aaggagtact cgcggacggt gtacatgctg
1441 tcctacctgg actcgcacgt cgacccgagg ggttggctcg agtggaacgg cgccgacttc
1501 gcgctcaaga cgctcttcta cggcgagtac cagaaccagg gccccggcgc cagcacggcc
1561 ggccgcgtca actggcccgg gtaccacgtc atcacggacc agagcgtcgc catgcagttc
1621 accgtggggc agttcatcca gggcggaaac tggctcaagg ccaccggcgt caactacaac
1681 gaggggctct ga
我的引物是用PRIMER5设计的，PCR产物的长度约880bp，退火温度用的是59度，可是没有带，用61.5度但是带很糊但是糊的地方不是在880bp，用63度试了一下则没带。郁闷啊
试不知道怎么进行PCR的改进为好，请各位大侠指点指点，小弟感激不禁。
这是我的引物序列
5'CGC CGT GTT CTC CAC CTT G 3'
5'CGC CGC CTT CCT CCT ACT CT 3'
NM_190847. Oryza sativa (jap...[gi:34908777] Links
LOCUS NM_190847 1692 bp mRNA linear PLN 07-OCT-2003
DEFINITION Oryza sativa (japonica cultivar-group) putative pectin esterase
(P0415A04.26), mRNA.
1 atgggtatca tccaccgagg ggacagcaca gtgaactgca cgtggctgaa cgaaaaacag
61 aacgggtacc atctaccgac aggtcgatac attcaaaacg cctacccatg caaacttcta
121 tcgttcacgc tgctctcttt agccaccgtg ctctgcctct gcgcgggcgc cgccttcctc
181 ctactctccc cgacggccac gaacctctgc gcgagctccc ctgacccggc ctcgtgccaa
241 gccatcgtcg ccgacgccgt cctagcgtcc ccgcactccc acccgagccg cccggcgcac
301 gtcctccgcg ccatccttgc cacgtctctc gatcggcatg acgcggccgc ggaagcggtg
361 gcgggcatgc gccggcgcgc cagcgacccg aggcaccgcg cggccctgga ggactgcgtc
421 cagctgatgg ggctcgcgcg cgaccgtctc gccgacgcgg cgggcgcgcc cgacgtcgac
481 gtcgacgtcg acgacgcgag gacgtggctc agcgccgtgc tcaccgacca cgtgacgtgc
541 ctggacggcc tcgacgacgg gccgctgcgc gactccgtgg gggcgcacct ggagccgctc
601 aagtccctcg cgagcgcgtc gctcgccgtg ctcagcgccg ccggccgcgg cgctcgggac
661 gtgctcgcgg aggccgtgga caggttcccg tcgtggctga cggcgagaga ccgcacgctc
721 ctcgacgcgg gcgcgggcgc cgtgcaggcg gacgtggtgg tggccaaaga cgggagcggc
781 aagtacacga cgataaagga ggcggtggac gcggcccccg acggcggcaa gagccggtac
841 gtgatctacg tgaagaaggg ggtgtacaag gagaacctcg aggtggggaa gacgaagcgg
901 gtgctgatga tcgtcggcga cggcatggac cagacggtca tcaccggcag ccgcaacgtg
961 gtcgacggct ccacaacttt caactccgcc acgctagcct tgtcgggcga cgggatcatc
1021 ctccaggacc tcaaggtgga gaacacggcg ggcgccgaga agcagcaggc ggtggccctc
1081 cgcgtgagcg ccgaccgcgc cgtgatcaac cgctgccgcc tggacggcta ccaggacacc
1141 ctgtacgcgc accagctccg ccagttctac cgcgactgcg ccgtctccgg cacggtggac
1201 ttcgtgttcg gcaacgccgc ggcggtgctc cagggctgcg tgctcacggc gcggcgcccc
1261 gcgcaggcgc agaagaacgc ggtcacggcg cagggccgga ccgacccgaa ccagaacacc
1321 gggacgtcca tccaccggtg ccgcgtcgtc ccggcgcccg acctggcgcc cgccgcgaag
1381 cagttcccga cgttcctggg ccggccgtgg aaggagtact cgcggacggt gtacatgctg
1441 tcctacctgg actcgcacgt cgacccgagg ggttggctcg agtggaacgg cgccgacttc
1501 gcgctcaaga cgctcttcta cggcgagtac cagaaccagg gccccggcgc cagcacggcc
1561 ggccgcgtca actggcccgg gtaccacgtc atcacggacc agagcgtcgc catgcagttc
1621 accgtggggc agttcatcca gggcggaaac tggctcaagg ccaccggcgt caactacaac
1681 gaggggctct ga
我的引物是用PRIMER5设计的，PCR产物的长度约880bp，退火温度用的是59度，可是没有带，用61.5度但是带很糊但是糊的地方不是在880bp，用63度试了一下则没带。郁闷啊
试不知道怎么进行PCR的改进为好，请各位大侠指点指点，小弟感激不禁。
这是我的引物序列
5'CGC CGT GTT CTC CAC CTT G 3'
5'CGC CGC CTT CCT CCT ACT CT 3'
