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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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liping121200

铜虫 (小有名气)

引用回帖:
3楼: Originally posted by 东乐乐 at 2015-10-19 09:05:11
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!...

这是我查到的,感觉蛮好,你看一下,真的要分段,在中间找个酶切位点分成两段就好了,每段一千多,也就可以了。再多了实在没意义,后面还麻烦。注意这个酶切位点必须是在这2800bp中只有一个,而且在酶切位点前后设计引物比较容易。

比如说,EcoRI吧,那么P出来的结果是 5'- XXX......GAATTCXXX,和XXXGAATTCXX.....XXX -3',然后酶切链接测序去吧。

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好好学习,天天向上
12楼2015-10-20 11:02:17
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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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[基金申请] 国际合作可查了,中了面上 (EPI+1)(金币+50) +18 Ldrop2023 2026-08-26 18/900 2026-08-26 11:15 by cmrandy
[基金申请] 系统进不去 +4 yanglien 2026-08-26 5/250 2026-08-26 11:10 by wenfengw83
[基金申请] 国合可查了 +3 paperzjh 2026-08-26 3/150 2026-08-26 10:41 by LemmonTr
[基金申请] 国合现在查不到了吗? +10 chengyan1220 2026-08-24 20/1000 2026-08-26 08:57 by peasantsprig
[基金申请] 在坚冰还盖着北海的时候,我看到了怒放的梅花。 (金币+10) +6 ziyangfang 2026-08-25 9/450 2026-08-25 20:26 by huagongfeihu
[基金申请] 没有任何消息-是不是就凉了 +9 图啦图啦 2026-08-24 10/500 2026-08-25 11:59 by 南海小哥
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