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Thread: Designing Primers

  1. #1 Designing Primers 
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    Hi all,

    I'm new to this site and I would like to ask a very basic question about primer design. I have designed my primers already but I see that on some occassions people might add a restriction cut site to the start of the primer. Why would this be done? And also is it necessary when ligating to a plasmid and cloning into an organism like E.coli?

    I understand the significance of cutting the plasmid to linearise it prior to ligation but i'm not sure why you would need a cut site on the PCR product...

    Any help would be much appreciated


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  3. #2  
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    A "Primer" being an elementary level text on a given subject, what the heck are you talking about?


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  4. #3  
    WYSIWYG Moderator marnixR's Avatar
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    i doubt whether Deoxy74 is talking about text books - it's more like this
    unfortunately, i'm not in a position to answer his question
    "Reality is that which, when you stop believing in it, doesn't go away." (Philip K. Dick)
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  5. #4  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    Quote Originally Posted by Deoxy74 View Post
    Hi all,

    I'm new to this site and I would like to ask a very basic question about primer design. I have designed my primers already but I see that on some occassions people might add a restriction cut site to the start of the primer. Why would this be done? And also is it necessary when ligating to a plasmid and cloning into an organism like E.coli?

    I understand the significance of cutting the plasmid to linearise it prior to ligation but i'm not sure why you would need a cut site on the PCR product...

    Any help would be much appreciated
    dunno. We never put a restriction site on ours. Because we would never use it. Must be some personal preference. Maybe they prefer a more specific ligase action or something, but i wouldn't bother.

    think about why you need the primers. What you are going to do with them. And if you are going to need a restriction site. No point getting one if you don't plan to use it.
    "Kill them all and let God sort them out."

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  6. #5  
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    Let's say that you have a vector with ----promoter---EcoRI---BamHI---- you want to clone a protein between EcoRI and BamHI, if the DNA protein sequence has the required restriction sites it's fine, else you have to introduce them artificially during the PCR step, designing the primers such that the end PCR product would be ---EcoRI----protein_of_intrest----BamHI---.
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  7. #6  
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    Hi Deoxy74,

    Restriction digestion enzyme sites may be used in order to generate an overhang, or 'sticky-end' in the product, in order to make cloning more efficient.

    BW,

    Tri ~
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  8. #7  
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    Ok excellent! Thanks for all the help guys!! So now I know that I need to incorporate these restriction sites on my primer if I want to ligate my PCR product to my plasmid. I'm assuming the same restriction site needs to be on the plasmid also so that they can ligate? So does this mean when designing the primers I will have to choose restriction sites that are on the plasmid also?
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  9. #8  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    check first how you lab ligates. Maybe they always do blunt end ligases.
    "Kill them all and let God sort them out."

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  10. #9  
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    Quote Originally Posted by Deoxy74 View Post
    Ok excellent! Thanks for all the help guys!! So now I know that I need to incorporate these restriction sites on my primer if I want to ligate my PCR product to my plasmid. I'm assuming the same restriction site needs to be on the plasmid also so that they can ligate? So does this mean when designing the primers I will have to choose restriction sites that are on the plasmid also?
    you have to check for compatible ends:
    Compatible Cohesive Ends, NEB

    compatible ends like well said by tridimity are "sticky ends". if you cut your vector and your PCR product with the same enzyme it works because the sticky ends left by the restriction enzyme (RE) are the same. Same enzyme is always compatible!

    You might want to cut your vector and PCR product to give e direction of insertion. If you cut with the same enzyme you possibly have your PCR product inserted in the other direction.


    as a side note:
    sticky ends are when the RE leave some nucleotide overhangs:

    EcoRI (the /backslash indicate the exact position where it cuts)
    1. Orginal DNA
    XXXXXG/AATTCXXXXXX
    XXXXXCTTAA/GXXXXXX

    2. after cutting:
    XXXXXG
    XXXXXCTTAA

    TTAA overhang -> can be coupled with an other DNA strand.


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