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2008116068

木虫 (著名写手)

[求助] PCR扩增出现的问题

我从一个8K质粒上扩增一段2.8K的目的片段,无论自己设计引物(16到22个碱基)还是按文献提供的引物(35和38个碱基),均扩增不出来。已经花了一个月的时间了,如果再扩增不出来,下个月就要换课题了。
目的片度的特点是GC含量约30%。
各位虫友,有什么好的建议吗?对我来说很重要,就麻烦大家了。

谢谢
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只要踮起脚尖,就离太阳更近一点!!!
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lovelyxixi81

金虫 (小有名气)

【答案】应助回帖

2008116068(金币+1): 2011-06-15 08:57:13
2008116068(金币+1): 2011-06-16 10:35:22
同意2楼意见,质粒测序。或者您把质粒序列贴上大家好判断。
6楼2011-06-14 23:29:19
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天使托

专家顾问 (职业作家)

T.Shen

★
amisking(金币+1): 鼓励应助 2011-06-14 19:00:24
设计引物扩增目的片段跟目的片段的GC含量有什么关系,只要引物设计没有什么问题我觉得应该没有什么扩增不出来的条带吧?

你何不把你设计的引物发上来大家给你看看是不是有神马问题。
Whenever,Wherever,Anyway!MustRemember:Fight!KeepFighting!NeverGiveUp!
2楼2011-06-14 17:48:11
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tanche163

捐助贵宾 (正式写手)

【答案】应助回帖

2008116068(金币+1): 2011-06-16 10:35:41
建议将质粒直接测序,看看是不是质粒有问题
阳光总在风雨后
3楼2011-06-14 18:41:43
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2008116068

木虫 (著名写手)

目标序列为
CTAACTTATAGGGGTAACACTTAAAAAAGAATCAATAACGATAGAAACCGCTCCTAAAGCAGGTGCATTTTTTCCTAACGAAGAAGGCAATAGTTCACATTTATTGTCTAAATGAGAATGGACTCTAGAAGAAACTTCGTTTTTAATCGTATTTAAAACAATGGGATGAGATTCAATTATATGATTTCTCAAGATAACAGCTTCTATATCAAATGTATTAAGGATATTGGTTAATCCAATTCCGATATAAAAGCCAAAGTTTTGAAGTGCATTTAACATTTCTACATCATTTTTATTTGCGCGTTCCACAATCTCTTTTCGAGAAATATTCTTTTCTTCTTTAGAGAGCGAAGCCAGTAACGCTTTTTCAGAAGCATATAATTCCCAACAGCCTCGATTTCCACAGCTGCATTTGGGTCCATTAAAATCTATCGTCATATGACCCATTTCCCCAGAAAAACCCTGAACACCTTTATACAATTCGTTGTTAATAACAAGTCCAGTTCCAATTCCGATATTAATACTGATGTAAACGATGTTTTCATAGTTTTTTGTCATACCAAATACTTTTTCACCGTATGCTCCTGCATTAGCTTCATTTTCAACAAAAACCGGAACATTAAACTCACTCTCAATTAAAAACTGCAAATCTTTGATATTCCAATTTAAGTTAGGCATGAAAATAATTTGCTGATGACGATCTACAAGGCCTGGAACACAAATTCCTATTCCGACTAGACCATAAGGGGACTCAGGCATATGGGTTACAAAACCATGAATAAGTGCAAATAAAATCTCTTTTACTTCACTAGCGGAAGAACTAGACAAGTCAGAAGTCTTCTCGAGAATAATATTTCCTTCTAAGTCGGTTAGAATTCCGTTAAGATAGTCGACTCCTATATCAATACCAATCGAGTAGCCTGCATTCTTATTAAAAACAAGCATTACAGGTCTTCTGCCGCCTCTAGATTGCCCTGCCCCAATTTCAAAAATAAAATCTTTTTCAAGCAGTGTATTTACTTGAGAGGAGACAGTAGACTTGTTTAATCCTGTAATCTCAGAGAGAGTTGCCCTGGAGACAGGGGAGTTCTTCAAAATTTCATCTAATATTAATTTTTGATTCATTTTTTTTACTAAAGCTTGATCTGCAATTTGAATAATAACCACTCCTTTGTTTATCCACCGAACTAAGTTGGTGTTTTTTGAAGCTTGAATTAGATATTTAAAAGTATCATATCTAATATTATAACTAAATTTTCTAAAAAAAACATTGAAATAAACATTTATTTTGTATATGATGAGATAAAGTTAGTTTATTGGATAAACAAACTAACTCAATTAAGATAGTTGATGGATAAACTTGTTCACTTAAATCAAAGGGGGAAATGACAAATGGTCCAAACTAGTGATATCTAAAAATCAAAGGGGGAAATGAGATCTATGGTAAGCCGATACGTACCCGATATGGGCGATCTGATTTGGGTTGATTTTGACCCGACAAAAGGTAGCGAGCAAGCTGGACATCGTCCAGCTGTTGTCCTGAGTCCTTTCATGTACAACAACAAAACAGGTATGTGTCTGTGTGTTCCTTGTACAACGCAATCAAAAGGATATCCGTTCGAAGTTGTTTTATCCGGTCAGGAACGTGATGGCGTAGCGTTAGCTGATCAGGTAAAAAGTATCGCCTGGCGGGCAAGAGGAGCAACGAAGAAAGGAACAGTTGCCCCAGAGGAATTACAACTCATTAAAGCCAAAATTAACGTACTGATTGGGTAGGCATGCTAATGCGGCCGCGAATTCCTGCAGCCCTGGCGAATGGCGATTTTCGTTCGTGAATACATGTTATAATAACTATAACTAATAACGTAACGTGACTGGCAAGAGATATTTTTAAAACAATGAATAGGTTTACACTTACTTTAGTTTTATGGAAATGAAAGATCATATCATATATAATCTAGAATAAAATTAACTAAAATAATTATTATCTAGATAAAAAATTTAGAAGCCAATGAAATCTATAAATAAACTAAATTAAGTTTATTTAATTAACAACTATGGATATAAAATAGGTACTAATCAAAATAGTGAGGAGGATATATTTGAATACATACGAACAAATTAATAAAGTGAAAAAAATACTTCGGAAACATTTAAAAAATAACCTTATTGGTACTTACATGTTTGGATCAGGAGTTGAGAGTGGACTAAAACCAAATAGTGATCTTGACTTTTTAGTCGTCGTATCTGAACCATTGACAGATCAAAGTAAAGAAATACTTATACAAAAAATTAGACCTATTTCAAAAAAAATAGGAGATAAAAGCAACTTACGATATATTGAATTAACAATTATTATTCAGCAAGAAATGGTACCGTGGAATCATCCTCCCAAACAAGAATTTATTTATGGAGAATGGTTACAAGAGCTTTATGAACAAGGATACATTCCTCAGAAGGAATTAAATTCAGATTTAACCATAATGCTTTACCAAGCAAAACGAAAAAATAAAAGAATATACGGAAATTATGACTTAGAGGAATTACTACCTGATATTCCATTTTCTGATGTGAGAAGAGCCATTATGGATTCGTCAGAGGAATTAATAGATAATTATCAGGATGATGAAACCAACTCTATATTAACTTTATGCCGTATGATTTTAACTATGGACACGGGTAAAATCATACCAAAAGATATTGCGGGAAATGCAGTGGCTGAATCTTCTCCATTAGAACATAGGGAGAGAATTTTGTTAGCAGTTCGTAGTTATCTTGGAGAGAATATTGAATGGACTAATGAAAATGTAAATTTAACTATAAACTATTTAAATAACAGATTAAAAAAATTATAAAAAAATTGAAAAAATGGTGGAAACACTTTTTTCAATTTTTTTGTTTTATTATTTAATATTTGGGAAATATTCATTCTAATTGGTAATCAGATTTTAGAAAACAATAAACCCTTGCATAGGGGGATCTCG
只要踮起脚尖,就离太阳更近一点!!!
4楼2011-06-14 22:44:11
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