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东乐乐

金虫 (初入文坛)

[求助] 用组织提取总RNA后逆转录得到的cDNA进行PCR一直未有目的条带,请大家帮忙! 已有3人参与

我打算做质粒构建,但是第一步PCR就进行不下去了。采集新鲜组织后立即用Trizol法提取RNA,用takara的逆转录试剂盒得到的cDNA,用师兄师姐留下的引物进行PCR可以扩增出相应大小的目的条带(约1500bp),但用我自己设计的几对引物都不能扩增出条带(目的条带大小:3564bp),只有最下面有疑似引物二聚体的条带,其余的一条带也没有。我想要获得该基因的CDS全长,之后进行细胞转染。选的酶切位点和载体都是实验室里现有的。实验新手有很多不懂的,请大家帮我想想办法,谢谢大家!

克隆载体:19T【T-Vector pMD19(Simple)】
表达载体:pEGFP-N1

引物:(其它引物跟这个差不多就是上下几个碱基的增减)
上游:5‘-CCCAAGCTTGCCACCATGCCTTTTGGTCTGAAGCTC-3‘[Hind III酶切位点,我引入了Kozak序列(GCCACC序列),据说这样有利于以后的表达]
下游:5’-CCGGAATTCTTAAATGAGTCTGGAGTTTTGGAGG-3’(EcoR I酶切位点)

体系:共25μl
高压过的纯水 16μl
10X LA PCR Buffer II (Mg2+ plus) 2.5μl
dNTP Mix 2μl
引物上游、下游各1μl
cDNA模板 2μl
Takara LA Taq 0.5μl

条件:35个循环
94℃ 2min(3min也做过)
94℃ 30s
55℃(58℃和61℃也做过)1min
72℃ 4min
72℃ 10min
4℃  ∞

CDS序列:共3564bp
ATGCCTTTTGGTCTGAAGCTCCGCCGGACACGGCGCTACAACGTCCTGAGCAAGAACTGCTTTGTCACACGGATTCGCCTGCTGGACAGCAATGTTATCGAGTGCACGCTGTCGGTGGAAAGCACAGGGCAAGAATGCCTGGAGGCTGTGGCCCAGAGGCTGGAGCTGCGAGAGACGCACTACTTTGGCCTTTGGTTTCTCAGCAAGAGCCAGCAAGCACGATGGGTGGAGCTGGAGAAACCTCTGAAGAAACATCTGGACAAATTCGCTAATGAGCCTTTGCTTTTCTTTGGAGTCATGTTCTATGTGCCAAATGTGTCATGGCTTCAGCAAGAGGCCACAAGATATCAGTATTACCTGCAAGTCAAAAAAGATGTGCTTGAAGGGCGATTACGATGTACATTGGACCAGGTGATTCGGCTAGCCGGCCTAGCTGTGCAAGCTGATTTTGGAGACTATAATCAGTTTGATTCTCAAGATTTCCTCAGAGAGTATGTGCTATTTCCTATGGATTTGGCCCTGGAAGAGGCTGTTCTGGAGGAGCTGACCCAGAAGGTAGCCCAAGAACACAAAGCCCACAGTGGAATCCTGCCAGCAGAAGCTGAACTGATGTACATCAATGAAGTTGAACGTTTGGATGGATTTGGACAGGAAATCTTCCCTGTAAAGGACAATCATGGAAACTGTGTACACCTTGGCATTTTCTTTATGGGGATTTTCGTGAGGAACAGAATTGGAAGACAAGCGGTAATATACAGGTGGAATGACATGGGGAATATCACTCATAACAAGTCGACCATTCTAGTGGAGCTCATCAACAAAGAAGAGACTGCCCTCTTTCACACGGATGATATCGAAAATGCCAAGTATATTTCTCGGTTGTTTGCCACACGACACAAGTTTTACAAACAAAACAAAATCTGCACTGAACAGTCAAATTCTCCACCCCCCATCAGACGCCAGCCCACCTGGAGCCGATCCTCTCTGCCCAGGCAGCAGCCGTACATCCTGCCTCCCGTTCACGTCCAGTGTGGTGAGCACTACTCGGAAACGCACACCTCGCAAGACAGCATTTTTCATGGGAATGAAGAAGCCTTGTATTGCAACTCTCACAACAGCCTGGACTTAAATTATTTAAATGGCACTGTCACCAATGGCAGCGTGTGTAGCGTTCACAGCGTCAACTCCCTCAACTGCTCGCAAAGTTTCATCCAGGCCTCCCCTGTATCCTCCAACCTCAGTATCCCTGGGAGTGACATCATGCGGGCCGACTACATCCCGAGCCACCGGCACAGCGCGATCATCGTGCCCTCGTACAGGCCAACCCCCGATTATGAGACAGTCATGCGCCAGATGAAGAGGGGGATCCTGCATACAGACAGCCAGAGCCAGTCTCTGAGAAACCTCAACATTATCAACACCCATGCCTACAACCAGCCAGAGGATCTGGTGTACAGCCAACCGGAGATGCGGGAGAGGCACCCCTACACTGTCCCTTATGGGCCACAGGGGGTCTACAGCAACAAACTTGTCAGTCCATCTGACCAGAGGAACCCAAAGAATAATGTGGTACCAAGCAAGCCGGGGGCAAGCGCCATCTCGCACACGGTGAGCACCCCAGAGCTGGCCAACATGCAGCTGCAGGGCAGCCATAACTACAGCACGGCCCACATGCTTAAGAACTATCTCTTCAGGCCACCGCCCCCCTACCCACGGCCACGACCTGCCACCAGCACCCCAGACCTGGCCAGCCACCGCCACAAGTACGTCAGCGGCAGCAGCCCGGACCTGGTGACCCGGAAGGTGCAGCTCTCGGTGAAGACCTTCCAAGAGGACAGCTCTCCGGTGGTTCATCAGTCTCTCCAGGAGGTGAGCGAGCCCCTCACGGCCACCAAGCACCACGGCACTGTGAACAAGCGCCACAGCCTGGAGGTGATGAACAGCATGGTGCGGGGCATGGAGGCCATGACGCTCAAGTCGCTCCACCTCCCCATGGCTCGCCGCAACACGCTCCGGGAGCAGGGACCGCCCGAGGAGGGGTCAGGCAGCCACGAGGTCCCCCAGCTCCCTCAGTATCACCACAAGAAGACCTTCTCTGATGCCACTATGCTAATCCACAGCAGCGAGAGTGAGGAGGAGGAGGAGGAGGCTCCAGAATCGGTGCCCCAGATCCCCATGCTCCGGGAGAAGATGGAGTACAGTGCCCAGCTGCAGGCGGCCCTGGCCCGCATCCCCAACAAGCCCCCGCCTGAGTACCCCGGTCCAAGGAAGAGTGTGAGCAATGGGGCTCTGAGGCAGGACCAAGCCAGCCTTCCTCCCGCCATGGCCAGAGCCAGGGTGCTGAGGCATGGGCCAGCCAAGGCCATCAGCATGTCTCGGACTGACCCGCCGGCTGTCAACGGGGCCTCTCTCGGCCCATCCATCTCGGAACCCGACCTGACTAGTGTGAAGGAGCGGGTCAAAAAAGAGCCTGTGAAGGAGAGACCTGTGTCTGAAATGTTTTCCCTGGAAGACAGCATTATAGAGAGAGAGATGATGATCAGGAATCTAGAGAAGCAGAAGATGGCAGGCCTGGAGGCACAGAAGAGGCCGCTGATGTTGGCAGCATTGAATGGGCTCTCGGTGGCTCGAGTCTCAGGGCGGGAAGAGAATCGAGTTGATGCCACCCGGGTTCCCATGGACGAGAGGTTCAGAACCCTGAAGAAGAAACTAGAAGAGGGAATGGTGTTCACAGAATATGAGCAAATTCCAAAGAAAAAGGCGAATGGCATTTTCAGCACAGCAGCTCTGCCAGAAAACGCCGAGCGCAGCCGAATCCGTGAAGTTGTCCCCTATGAGGAGAATCGAGTAGAGCTGATACCAACCAAAGAAAATAACACAGGATACATTAATGCCTCCCACATCAAGGTGGTGGTTGGCGGGGCAGAATGGCACTACATAGCCACCCAGGGGCCCCTGCCACACACGTGCCACGACTTCTGGCAGATGGTGTGGGAGCAGGGAGTGAATGTGATTGCCATGGTCACTGCAGAGGAGGAGGGTGGACGAACCAAAAGCCACCGATACTGGCCCAAACTAGGTTCAAAGCACAGCTCAGCCACCTATGGCAAGTTCAAGGTCACCACGAAGTTTCGAACGGATTCTGTTTGCTATGCAACCACGGGCTTGAAGGTCAAGCACCTTTTGTCTGGGCAAGAAAGGACGGTGTGGCATTTACAATATACTGACTGGCCAGATCACGGCTGTCCAGAAGATGTCCAAGGATTTTTATCCTACTTGGAGGAGATCCAGTCGGTCCGTCGCCATACCAACAGCATGCTGGAAGGCACCAAGAACCGGCACCCGCCCATCGTGGTCCACTGTAGTGCTGGGGTGGGAAGGACCGGCGTGCTCATTCTTTCTGAGCTGATGATCTACTGCTTGGAACATAACGAAAAGGTGGAAGTGCCCATGATGCTGAGGCTCCTCAGGGAGCAGAGGATGTTCATGATCCAGACTATCGCTCAGTACAAGTTTGTCTACCAAGTCCTCATCCAGTTCCTCCAAAACTCCAGACTCATTTAA


总的mRNA序列:其中654bp至4217bp为CDS区域
ACTCGCACGGCCCCTTCCTCCCTCCTCTCCCGGCCGCTCGCATTTCCTGCCGCTCTGGCTCTCCCGGCCCCTCAAAGTTCTTTCCAACTTTTTCTCGGCGGAGTGAGCGCAGCGGGCGCAGACTCGGGGGCAGGTTGCTGTGCTTCTCCGGGCTCAGCCGCCTGCTCTCCTGGCTCAGGTCCTCGGGGAGCCCTAGACAGACATCAAGTGGCCACTGGCGCTCCTTCCCCTCCCAGCTGAGCCATCCTCCCCGGCCTCCTCGGGCGGGACAGCCCCGTGCTTAGGTTTTTCTCCTTTTCTCCCCCGGTGCGCCTCTGCTCGGACTCTCGCGCCGGGATCGCGGCGGAAACCTCCCTCCCCTTTCGCCTCCTGCGGCTCCTTCCCTTCGCCCCTCCTCCGCCAGTCACTGGAATCAATTCCGTGGGGAATCGGCTCCGCCGCCGCGAAGGACAGCCTTTCCGCGCGGGACTCCGGGGCGCCACGGGGGCCATGTAAGCAGCTATCTTCCAGAGGGCCACACTGGGCATGGACACCCTTTTCCCTGCCTGGAGGAGCACAGGTGATAGTGTAATTTTCCAGTCACGAAACTGCTAAGGCCATCTCAGGGGCGTGTGCGCCAGGATAGGCGGGCGGCGTCCGAGGACCACATAGCCATGCCTTTTGGTCTGAAGCTCCGCCGGACACGGCGCTACAACGTCCTGAGCAAGAACTGCTTTGTCACACGGATTCGCCTGCTGGACAGCAATGTTATCGAGTGCACGCTGTCGGTGGAAAGCACAGGGCAAGAATGCCTGGAGGCTGTGGCCCAGAGGCTGGAGCTGCGAGAGACGCACTACTTTGGCCTTTGGTTTCTCAGCAAGAGCCAGCAAGCACGATGGGTGGAGCTGGAGAAACCTCTGAAGAAACATCTGGACAAATTCGCTAATGAGCCTTTGCTTTTCTTTGGAGTCATGTTCTATGTGCCAAATGTGTCATGGCTTCAGCAAGAGGCCACAAGATATCAGTATTACCTGCAAGTCAAAAAAGATGTGCTTGAAGGGCGATTACGATGTACATTGGACCAGGTGATTCGGCTAGCCGGCCTAGCTGTGCAAGCTGATTTTGGAGACTATAATCAGTTTGATTCTCAAGATTTCCTCAGAGAGTATGTGCTATTTCCTATGGATTTGGCCCTGGAAGAGGCTGTTCTGGAGGAGCTGACCCAGAAGGTAGCCCAAGAACACAAAGCCCACAGTGGAATCCTGCCAGCAGAAGCTGAACTGATGTACATCAATGAAGTTGAACGTTTGGATGGATTTGGACAGGAAATCTTCCCTGTAAAGGACAATCATGGAAACTGTGTACACCTTGGCATTTTCTTTATGGGGATTTTCGTGAGGAACAGAATTGGAAGACAAGCGGTAATATACAGGTGGAATGACATGGGGAATATCACTCATAACAAGTCGACCATTCTAGTGGAGCTCATCAACAAAGAAGAGACTGCCCTCTTTCACACGGATGATATCGAAAATGCCAAGTATATTTCTCGGTTGTTTGCCACACGACACAAGTTTTACAAACAAAACAAAATCTGCACTGAACAGTCAAATTCTCCACCCCCCATCAGACGCCAGCCCACCTGGAGCCGATCCTCTCTGCCCAGGCAGCAGCCGTACATCCTGCCTCCCGTTCACGTCCAGTGTGGTGAGCACTACTCGGAAACGCACACCTCGCAAGACAGCATTTTTCATGGGAATGAAGAAGCCTTGTATTGCAACTCTCACAACAGCCTGGACTTAAATTATTTAAATGGCACTGTCACCAATGGCAGCGTGTGTAGCGTTCACAGCGTCAACTCCCTCAACTGCTCGCAAAGTTTCATCCAGGCCTCCCCTGTATCCTCCAACCTCAGTATCCCTGGGAGTGACATCATGCGGGCCGACTACATCCCGAGCCACCGGCACAGCGCGATCATCGTGCCCTCGTACAGGCCAACCCCCGATTATGAGACAGTCATGCGCCAGATGAAGAGGGGGATCCTGCATACAGACAGCCAGAGCCAGTCTCTGAGAAACCTCAACATTATCAACACCCATGCCTACAACCAGCCAGAGGATCTGGTGTACAGCCAACCGGAGATGCGGGAGAGGCACCCCTACACTGTCCCTTATGGGCCACAGGGGGTCTACAGCAACAAACTTGTCAGTCCATCTGACCAGAGGAACCCAAAGAATAATGTGGTACCAAGCAAGCCGGGGGCAAGCGCCATCTCGCACACGGTGAGCACCCCAGAGCTGGCCAACATGCAGCTGCAGGGCAGCCATAACTACAGCACGGCCCACATGCTTAAGAACTATCTCTTCAGGCCACCGCCCCCCTACCCACGGCCACGACCTGCCACCAGCACCCCAGACCTGGCCAGCCACCGCCACAAGTACGTCAGCGGCAGCAGCCCGGACCTGGTGACCCGGAAGGTGCAGCTCTCGGTGAAGACCTTCCAAGAGGACAGCTCTCCGGTGGTTCATCAGTCTCTCCAGGAGGTGAGCGAGCCCCTCACGGCCACCAAGCACCACGGCACTGTGAACAAGCGCCACAGCCTGGAGGTGATGAACAGCATGGTGCGGGGCATGGAGGCCATGACGCTCAAGTCGCTCCACCTCCCCATGGCTCGCCGCAACACGCTCCGGGAGCAGGGACCGCCCGAGGAGGGGTCAGGCAGCCACGAGGTCCCCCAGCTCCCTCAGTATCACCACAAGAAGACCTTCTCTGATGCCACTATGCTAATCCACAGCAGCGAGAGTGAGGAGGAGGAGGAGGAGGCTCCAGAATCGGTGCCCCAGATCCCCATGCTCCGGGAGAAGATGGAGTACAGTGCCCAGCTGCAGGCGGCCCTGGCCCGCATCCCCAACAAGCCCCCGCCTGAGTACCCCGGTCCAAGGAAGAGTGTGAGCAATGGGGCTCTGAGGCAGGACCAAGCCAGCCTTCCTCCCGCCATGGCCAGAGCCAGGGTGCTGAGGCATGGGCCAGCCAAGGCCATCAGCATGTCTCGGACTGACCCGCCGGCTGTCAACGGGGCCTCTCTCGGCCCATCCATCTCGGAACCCGACCTGACTAGTGTGAAGGAGCGGGTCAAAAAAGAGCCTGTGAAGGAGAGACCTGTGTCTGAAATGTTTTCCCTGGAAGACAGCATTATAGAGAGAGAGATGATGATCAGGAATCTAGAGAAGCAGAAGATGGCAGGCCTGGAGGCACAGAAGAGGCCGCTGATGTTGGCAGCATTGAATGGGCTCTCGGTGGCTCGAGTCTCAGGGCGGGAAGAGAATCGAGTTGATGCCACCCGGGTTCCCATGGACGAGAGGTTCAGAACCCTGAAGAAGAAACTAGAAGAGGGAATGGTGTTCACAGAATATGAGCAAATTCCAAAGAAAAAGGCGAATGGCATTTTCAGCACAGCAGCTCTGCCAGAAAACGCCGAGCGCAGCCGAATCCGTGAAGTTGTCCCCTATGAGGAGAATCGAGTAGAGCTGATACCAACCAAAGAAAATAACACAGGATACATTAATGCCTCCCACATCAAGGTGGTGGTTGGCGGGGCAGAATGGCACTACATAGCCACCCAGGGGCCCCTGCCACACACGTGCCACGACTTCTGGCAGATGGTGTGGGAGCAGGGAGTGAATGTGATTGCCATGGTCACTGCAGAGGAGGAGGGTGGACGAACCAAAAGCCACCGATACTGGCCCAAACTAGGTTCAAAGCACAGCTCAGCCACCTATGGCAAGTTCAAGGTCACCACGAAGTTTCGAACGGATTCTGTTTGCTATGCAACCACGGGCTTGAAGGTCAAGCACCTTTTGTCTGGGCAAGAAAGGACGGTGTGGCATTTACAATATACTGACTGGCCAGATCACGGCTGTCCAGAAGATGTCCAAGGATTTTTATCCTACTTGGAGGAGATCCAGTCGGTCCGTCGCCATACCAACAGCATGCTGGAAGGCACCAAGAACCGGCACCCGCCCATCGTGGTCCACTGTAGTGCTGGGGTGGGAAGGACCGGCGTGCTCATTCTTTCTGAGCTGATGATCTACTGCTTGGAACATAACGAAAAGGTGGAAGTGCCCATGATGCTGAGGCTCCTCAGGGAGCAGAGGATGTTCATGATCCAGACTATCGCTCAGTACAAGTTTGTCTACCAAGTCCTCATCCAGTTCCTCCAAAACTCCAGACTCATTTAATCACCCCAATCCAGCTCCTGGAGGAGGGACCCAGCTCCATCGCGCTGGAGGAGAGTCACCTCCAGACAACATCTGCTCCCCCCACAGGGGTGCAGGTGGCTGGCAGCAAACAGGCTCTCTGAAGACAGTAGCCAAGATTATTCACACATACCATGTATTATTTTATATGAGATAATTTATTTTTTTCCCCTTTGGAATAACTTTTGTGAATTATTATAATGCAGTTTCCCTAGTAATATAGTACTTTTCATTTGAACCACATCTTGACTGATCTGTATTGTAATATATGTCAGCAGGTAAGGTTGCCTGCTGGATCATTTTGAGGACAGAGGCATGAGGGAGCACATCTCTTGTGAAGTTGCAGCCAGATTTGTAACCAACCCTGAAATTCATCAGCTTAATTCATTTATCAGCTTGATTCATTCATCATTCATTGCTTATATCCAAAGCAAAGACGGTAAGAAAATGAATTCATCCTGAAATATAAAGAAAAGGGTCTGAAGGAACAAACACGATTCTCTTATATTTTGGGGCTCATGAGCCTTGATAGACAGTTTCCTCTCGTCTTCATTTCCACCCCTCATCCTCAGTAGTCTCCTCTCCCCCACGCCCCACCCCAACTTCCCCCCCAAGCTTGAGTTAAAGACAGAATAGCTAAAGACAGTGCTGCCTTTACAATGCAGTAATTGCCATCTTTGGGGCCGAAAGACAAGCTCTGTGTTGTGCTTTTCTTGACCACCCCTTATCCTGGGCTCTGGAGCTTGTGTTTCCCTGCTGGCGACTGTACCTTGGGTATTTGTTGCTACCTCTCCTGTTTGCTCAGTAGGACCCTGTCTGGTGGCATTGAGGCTCTGGACCAGACCATCTGTGCAGTTAAGGCTCTACCCTGATTGAGAGAGGATAGCAGACCTAGAAAGAGAAAGGAGTTGGGCAGGGCCTTTGAGGATTGTGTTTTTCAGGCAGGGCCTTGATGATCATTGTTTTTTATTTAAATAAGATGTGTGTGCTGGACAGAGACCTAAAAGTTGAGGTCACTAAGTCATTGGAAAGGCCATCAAGGAAACAGATGGGGAAGCTGATTTATGGGAGCTGTAAGGCATTTAGCTACATAACAGGGGTCCTGGCCAGGAAACACATCAAATGTGACCCCCGCTGTGCTGATATCATCTTCAGGCTTTGGTCTGCAAGATCAGAATTAATCCCACTCGGGACCCCATAGTCCAAACTTGGGGCCACTTGATGAACGATGGTAGAATTGTCATTGGCAGAGCCCTGTGCTTCTTTCCTTTTCTTCATAAAATCCACTCGCTGGTCAGTTATCTTCACTTTGAAGCCCAGTTCTTAGTTTCTTCCTATGGCTTCATTGGTCAGTGTCCTTCTGAATTTCCAAGGATGGTACACAATAAATCATGTTTTGTACTTTTTTCCTCTTACTGCATTTTGGGGGATTTATCATTCTATGTCTACCTTTTCTTGAGTACAGCTTTGATATGCACCTGTTGTTACGTGGTGATGGGAAGTCACAGGCGTGCTCTTTCTAGTTAATTTGATGCCACATCTTCCTTGTCTTTTCAGCTTGGGAAAAAGGCGGCAGTGGAGGAAGGCATGGAATGCCCACAGTGGTCAGTTCAAAGAACAAACGTGCAATTAAAAAACTGTAGTCAGCCAGGCACGGTGGTTCACACCTGTAATCCCAGCACTTTGAAAGGCCAAGGCGGGCAGATTGCTTGAGCTAAGGAGTTCGAGACCAGCCTGAGCAACATGATGAAACCCCGTCTCTACAAAAAGTACAAAAATTAGCCAGGCGTGGTGGTATGCCCTGGTAGTCCCAGCTGCTCGGGAGGCTGAGGCAGGAGGATCACCTGATTCTAAGAATTCGAGACTGCAGTGAGCCGTGATCTTGCCACTGTAGTCCAGGCTGGGCTACGGAGAGACCCTGCCTCCAAAAAAAAAAAAAAAAAAGGAAAAAAGGTTGTCAAGAAAAACTAGATGTTAGGAGAAAGGAAAATTTAATTGCAGTTTTTTTTCTTAGAATTGACTGCTGTGAGAGTTCCATATGCCTTTCTTCATTGCTGCTTTTGTCCCCCGTGAGCTAAAAAGATGGAGTGACATCAAATCAACCAGAAAAAGTATGCCTTTGTGACATCCCATCACCACATGCCAACAGGTATATATTCCCCATTAAGTTCTTCGGAATAGGAATCCTCTGTTTCAACCTGGCCAGGTGTTGTGGTGGCTGTACTCTAGTTAGACTCGGAATATCTGGGGATGGAGGGCTTCCCCTGTGTCTTCTACTTCAAGGTCTGAAGGCTCAGTGAAGGAGTATAATCTGCTGATCTTTGTAGATTCTGGAGTTTTGTTGTATGTCCTGGAAAGAAACCCATTAGTATTACATGTATTTTCAGTGAACAGAGCTTATAACCCTTATTATAAGAAGCTCATCAATAAGCAAAAAGATACTTGTTTCCTTTCCTTGGAGGTTTTTCCATCCTTGGGATATTCTGCTGTTAGGGATGTTTTAGCAAGTGGTCTCAGTTACTGGTTTATTGCGTGATGAACAACATCAGTATTTATCTTTTATCTCTAAGCCCCAAGGTGGGCACTGTTAGAATATGTCTCATGTGGACAGCATATAGATCTGGTGCGTCTTTGAGGTCGTCAGAGCTCATGGGCTTCCCTGAAATTCATCCACTGTCCCTGCCGTATGCTACGGGAATATTCATTAGTGTACAAAATGCAGGGAGGAAGTAGGTTTAATATTCAACTTTCTAGCCAAAGTTTATATTGAAACCCAAAAGAAAACATTTAAGAGTTGTTCCACATATTTCACTTTTAAAAACAAATGCCTTTGGTTCTTTAGCACATTTTGCATTCCTTTTCACATCTCCAGTAAATGCCAACATATCTCCTGTTAAATTAGCAGCAGCCATTTAAAGTCCTTTCGGTGGCATCTGCATAATAATTGCCCAGAGATGCTTTATATCTGGGAAGCAAGCCAAGGAATAAACCTTGAAGCAAAGTGTATTAAATTAGTTATCTAGTTAGAGCTTTTGGAATGATTTCCTGATGATGTATCAAGTCTGAAGCTGGAGCTGTCAGTGTCTATTGCTGCAGTTTGGATTTGAAGGGAGAAAATGTAAAATGGAGGAAAAAAAAGTTACCATCTCACAACAAAGCCATCAAACATTTTCCAGCCGCTGTTTTCGAGGTTTTCCAGTTGAACTGTTTGGTTTCTTTCATCCACACTCATTTGGATACATTGACCCGAGGTATTCATCCTTGTTTACTGTGGTCCCTGAATCATGGGGGCTGAATTTGATGTCTTCATCCTTGAGATGAGCCTGCTGGCTTAGCTGAGGAATGTCCTGCTGAGGTTTCTTAGGTTTCCTTGGGTTCTAAGGATATACTGGATATACCATCTTTTAGCAAGAGTATCTGGTAGCATTTACAGATAGCATAGACATTGGTATGCACTTCTTTCCCCAGATAGGAAGTAAAGGAGGATTTAGTTGCATGAAAAAAGGATGTTAAACATTGATTACATAGGAGTAAAGATGAATGAGCTGCAATATTCAGTCGGAGCTAAACAATAAGATCAGGGAAGGTAAAAATACCTATGTGGAATATTTTGAATCGTAAGCTTTTGAGGAGCTTAAATTGAGAGAATTTTACTTTTAATTTTGTAGATTGAGAAGAGGAACCGCTTTTTAAAATTATAGCTAAACTGTCATTGTTTTCCTAAGAGTCACTTGGCCATCTCTGGCCCCCTCTTTCATCAGCCTGAAGAGAGGGTCTTTGTAGACTGCTGAGGGTGGGCCTTGTAGGACTTGACCATGGCTTACACCTACTTAACCTTTATCCTGCTTTCTTTCAGCTTGTGCTTTTCAGTTATAAACTCCAGTGGGTACAGCAGGCTGGCCTTTTCATCCAGCTGATTATTTTTCCAGCTTAATATAGATTGACCCATATGAAATTTCCAATAATGGACCATATTTTCTGCAAATAGACAGTACTCGCATGGATCACCTATATCTTCCCCCTGATACACTGTGGGTCCCAACACCAGATGTCATTTCTCCAGAGCAGTGCTAATGAACACAAAAGGTATACCCTGGGTGGCCCAGCTCTTTTCACGAACGTGCTGCCCTGCTCATAGTGATCCTTGCATCACTTGGTGAATGGGCCATCTCCTGGGACATGGAAGTTGCAGAGGTAGATAGTGCACTGCAGCTTCTCTTAAGCCGGATTGGCCATCAGGCATATCACTCTGGAGTTTTTAGCTGCTGTCCTTTCCCGATGAACAGTCTGTATTAGCTGACCTCAGCCTACTTGTTACGTGACGTATGGGTCCCAAAAGTGTCCTTTGTCAAAAAGCAGAATGTGCCTTCTAGTCTCCCTTTCCCCATCTAATGGTGTATTCGATGGTGAAGATGAGTACAGTTGACCACCCCTATCTGTGGGTTCCACATCCCTATATTCAACCAAGTACAGATTGAAAAATATTTGGGGAAGAGGGGAAACCCACAAAGTTCCAAAAAGCAAAAGTTGAATTTGCCACATGCTGAATACTACATTGAATTCACACAAATGAAGTGATGTGTAGGCATTGAGTTAGGTATTGTAAATAATCTAGAGATGATTTAAAGTATGCAAGAGATGTGCATAGGTTATATGCAAATACTGTGCTATTTTATATAAAAGACTTGAACATCCATGGATTCTGGTATTCTCAGAGGGTCCTTGATTGCCCCCTTTGGTAAAGGACAACTATTTCGTTACTGATTTTCGTTTGGGGAAGATCTGTCAATCCCTTGAGGTGCGGGGGTTGGGGGGATGGAGGGTACAGGGCATTCTAGGATGTGTGCCAGGGAGCACAGATTCAAGGGATGGGATTGAGTCAGACCTGTGTTCTTACTCAGTGTCAGAAATAACTCTGTGGAGCTCCTAGAGTAAGACATTTCCGGAAGCACCACATAATTACTGTTGGGCTCTTAGGGTAGCCCTTTTAGGGAATTGAGCATTCCCATGTTTTACCAACAATTATTCTGCTGCTGTGTTTTATTATATTGCCAATGGTTTTGAGACACTCATCATGCTCTTATTTAGTGATTTCTTTTCATGAGCAGAGCAACAGCTCATCCAGCATGGTTTCCAAATGGAGAAATTTGGGTCTTCTGTAGAAACCACACAAATTCTCCAATGGCCTACAGCCTTATGGTTGGCACACTAGTTGGCCCTATAGGGTGGAAATAAAGCTGTAAGATGTTAAACTGCATTTGATACTTCTCTTGAACGCTGAGCAAGGAAGCAAAATAGTTCTTGTCTTTACTTAAGCTTCTAAGACATTTTGGGGCAAAGGACCTTACAGATGGCGTCTGTTGAAAGTAACAGCAACGTGCCAGGGAGAAATGTGGGGGAAATCTCATCAAATTCTGCCACCTCAAATGTGTTGTCCAGAAGTCAGTGTTATTCAGGGGGCCCTGTGAAACTTGACCACCAGCTTGCGCCATCAACACTCAGCCTTTATCCAGCCTGCTCTCAGCTTTTGTGTTTCTGTTAGAAACTCAGATAGGTAAGTATTTTTATTCAGTAGACTACCTTTCTTATCTTTTCAGCTTAATATAGCTGCATCTTCTTTCTCAAAGCCAAACTAAGATATTCTCTATTAAAATGTCCATGAGCCTAGCATTGAGTGTCTGGCATCCATCAATTTCATAGACTGGAAAATGATTGTTGTTTGGTACAGTAAAGAAGAGGATGTGCATCAGTTCCTACCGTTTGCAGCTTTGTTTTTAGCCTTCCCATTTTAAAAATAAATTCAAGGACATTGAGTTACAAGGCAGGAGGGCTGGAGCTACTGGGCAGCCTGAATATGACAAGCATTTGTGTGGAAAGTCATTGCTCCTTCTGCCACACTTTGGGCCATCAGGATCATTCTTTCCCAGAAGTGCCATAAACTTGCTCAAAAGTTTCTATAAATGGGAAGAGGGAGAGGAAGGATTTTTGCATCAGTCCTGAAGTTGCTATCCAAAAGTTCTCTGTTTCACAAATAATTTTCTGAACTCTGGAATGCCTCTCCTACTCCCTGCCTCCCTTTCTGTAATGTCAGAGTGATGGAAACCACCAAGTGGCATGCTAGGGAAAGCCTGCAGCAGTGTTGGAGTCTATTTCACCCTAGCTCATAGTTTTAAACTGTCTTCACTGTTGAGGTAGAGCTTGATGAATGTCATGGATTATGATGTGTGGTTTATCATATTTGCCTGGATTTGCTGATCAAAAGCACCATCTTCCCTCGCCTGCTGCTGGCAGCCTTTCCTTGCCTTGCTTGTTAGCAGAGCATTCTGCTTACCCATGTGGCTCCCAGAGTTAGCAGCCCCGGCTCTTGGATTTCTTGATTCTTCTCCCCTGTGATCTCAGAGGTGCTGCAGAGGACATTCCCCTTTAGAGCAAGTCATGTTTCTATTCAGGCCACAAAACTGGGATGTACATGCAGTGACTTTGGTGTTCCTTGTCTTGTTCAGGGGAACGGGTGGGACTGTTGTGTGCTGTCACCCTCTTCATTCCATGAGCACCTTGTTCACTTAGGGTCTGCTGCCTTTTTTTTTTTCTTTTTCTTTTTTTTTTTTTTTTTAGTTTTTGAGATCGTGTCTCACTCCTGTTGCGCAGGCTGGAGTGCAGCCTCCAAAGTAGCTGGGATTATAGGCGTGCACCACCACACCCGGCTAATTTTTATATTTTTAGTAGAGACGGGGTTTTGCCATGTTGGCCAGACTGGTCTCAAACCTCTGACCTCGTGATGACCCCCCTCGGCCTCCCAAAGTGCTGGGATTACACCCAGCCTCTGCTGCCTTTTCATATTTCCCCATCTGCTTTATGGATCAACTCTCAACAGTACACTTTTTCTTTTCTTTACCTACCCTATGAGTGCAACCCAGATGTAAGAGTTAATCCTCGTCAGAGAATCATTGCCTTAAACCTCTCAGAAATATGTAATTAGGAAATCTTATTTTAATTTTTTAAAAATTGCTTGTATAGTTTCAAAGAATAAGATCTGGCAAATGGCCAGATGTGGTGGCTCACACCTGTAATCAAACCAGCACTTTGGGAGGCTGAGGCAGGCAGATCACCTGAGGTCAGGCGTTCAAGACCAGCCTGGCCAACATGGTGAAATCCCGTCTCTACTAAAAATACAAAAAAAAAAAAAAAAAAAAAAATTAGCCAGGCATGGTGGCACATGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGATAATTGCTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCATGCCACTGCACTCCAGCCTGGGCAACAGAGTGAGACTTTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAGATCTGGCGGATGAAAATAACCAGAATGAAAATAGCTAGAAAACTCAGCAAGCAGGAAGCTCCCTTTCTCACCCTTTTGTTCCCTTGCCGATAGAATCAGTCACTATTAGAAAAAATGAAAGACGCTCTGTTTAAAACAATGATGACAGCAGTACTTAATATGTATTTCGAGGTGAACTTATATAGATTGAGAGAGGCTGCATTTGGCAGACTGATGTATAGGAAGACCCATTTGTTTCTAGCTTCTCCCTGCAGGGAAAATGCTTTCGTCATTATAGCCTCTTTACACAGACTGGCCATTCTAGTGAAACAGGTGGTAAACCTTTGGGCTGCCCAGAAACATTTTATCTGTTTTCACTTACCTAGGAAGGGGAAAGATTAGCGGGTCATCCAAAATCTGTATGTAAGCTATCTTCATTTTCTTCCCCAACCTTCTCCTCCTGGGAAACACAAATGCTATCTCATCTGACAAAAGGTTTTAGAGGATAAAGCTGAAAAGATTGGATTGGGATCTTTTTGTGGCTTGGGGCGGAGCCTTTTGCTAAAATCTCAAGAATGCTGCTTTGAGTTTAGCTAGGGTGGCTCTCAGAACTGGGGTGCCTGGCATTCTCAGCATTTCTCAGGGGCCTCCCACCTCTGACAACTGCAGTGTTAGCTAATACATACCTTGAGCATAGAACTGAATGCTGTAATTCAGAGCCATTTTTTTTTTCAACTTGAACATTGTACAATTTTACTGCAATTTCCTTTGAACTTTCTTGCCACTGTTTGGAATCTTAAAAATTCATTAGCCTTCTCCTTTCTGACATAAAGCTACTCTTCATCAGAGATGAGTTCCTATGTATGTCCTTTGTTCCTTCAATAGCTAATTAATGTGCTTGAGGATACTTCAGTGGAAAAAAAGGTTTAAATATGCAAATTACTAATAAATGTGTAACCTTATGTAACTTGTGTTACATCAAGTAACAAGCTAATCTAGTTTGTTTCACTGGACTAGGCTTGTGCTCCCTACTTCAGTATTTTGATGCTTTCCTTGATCTTTGTTTCACAAAATGTTGTGAATTTTGGTATCATTCAAAACAAATGACATTTATTAGGTTTCATTTTGAAACGATGTACAGACAAGTCCCCAACTTAGAAACCGGTTTGTTCTTAAGGTTCTTGCGTCAGCCCATAGAAGCCCACTGACCTCCACCACAGCCCAAATGGAGGGCTGTGATAGCCAGATCTGGTTGGCTTTTGTGGGCTGACCCAGACATTTAATCACCATCTCTTATGTTGTTGCCGTAAGAAATGCATTCCAGGTTGGGACTTGGGATCCTGAGAGCACATTCGCCCCCTGTGGTGGCCGCTTGCCACCTTGCAAGATGGAAGCCCAGTCTCCTTACTACCAAACTGTAGTTGTAAGCAGAGGGAGGGGTGAGATGTTTATAGGACATTCCCTAAGCTGGGGAGTGATTTTTATCACTATTCATGTCAACTGTACTTTGGTATAGACTCCCTATCAATTTAATAATATGAAAAGCCTAAAATAAAACTATGCATGCTATTCTATGTGCTATTTTATATCAGTAAATAAGCTTATGCTTGCCAGTTGTATACACAGTTATGAGGTGTATAGAACTGACTTTGACAGTATTTTTTGCACTGTTTCCTATCTGTTTTTATAAAGTCTTATTTAGATATTGGACCTTGTTGATGTTCTCACTGCCCTTGTGCTTGCTATAAAATGTTTCATATGTGCCTTTACAAATGTGAGATCTTTATTCTAACCTTTTTTTGTAAAAGATATCTATTGATTTCCATATGCAATAAACCTTTTTTTCAGAGAAAAGTTA
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董博士

金虫 (正式写手)

【答案】应助回帖

目的片段太长了,超过3Kb就算P出来也容易发生部分位点突变,这种情况还是建议分段PCR,然后连接,这种原理就是利用粘性末端连接的,每段的引物都设计的带一点下一段的前5-10个碱基

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医学&统计学
8楼2015-10-19 17:43:42
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liping121200

铜虫 (小有名气)

【答案】应助回帖

感谢参与,应助指数 +1
你的目的条带太长了,一般情况下PCR扩增的最大片段就是3kb,你需要想其他办法了,用扩长片段的long taq酶或者做分段克隆做拼接了

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好好学习,天天向上
2楼2015-10-16 15:45:27
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东乐乐

金虫 (初入文坛)

送红花一朵
引用回帖:
2楼: Originally posted by liping121200 at 2015-10-16 15:45:27
你的目的条带太长了,一般情况下PCR扩增的最大片段就是3kb,你需要想其他办法了,用扩长片段的long taq酶或者做分段克隆做拼接了

谢谢你的回答,请问如果做分段克隆的话有什么好的建议吗?例如酶的推荐和引物设计的经验能分享一下么?因为没有接触过分段克隆,请多多指教!
3楼2015-10-19 09:05:11
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秉秉的法兰西

新虫 (小有名气)

我们一般会先p个内参看看,先确定cdna没问题

发自小木虫IOS客户端
4楼2015-10-19 12:02:43
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