123321 asked in 科學生物學 · 2 decades ago

生化問題PCR

How to create an EcoRI site (CTTAAG) for a desire DNA fragment by PCR?

Update:

可以用中文嗎? 謝謝~

2 Answers

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  • C C
    Lv 6
    2 decades ago
    Favorite Answer

    If you want to create EcoRI sites flanking the ends of the DNA fragement (I assume you are trying to clone this DNA fragment you're trying to amplify), simply construct your primer to fit the DNA fragments you want to amplify, and then tag on the EcoRI site at the end of the primer. This way the primer will still anneal to the target, but after the second round of PCR all the elongation step will start incorporating the EcoRI site.

    After you get your PCR products, you can then digest the product with EcoRI, and then ligate it into the plasmid.

    2005-12-15 14:26:57 補充:

    I don't know why you claim it's not efficient, and insist on doing extra work through TA cloning. I've done this repeatedly in lab.

    2005-12-15 14:27:30 補充:

    I don't know the "efficiency" per se, but it works well enough that you'll get enough product to insert into a plasmid successfully. And once you get one in you are relying on the bacteria to amplify for you anyways.

    Source(s): I am a graduate student in genetics
  • Anonymous
    2 decades ago

    PCR product can't be digested directly by enzyme. The efficiency is not good. You have to do TA cloning first or to add few base more of the primer with EcoRI site.

    Source(s): I am a master
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