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    Hey guys, really struggling with this question:

    i) Explain the effect of different temperatures (used in PCR) on the DNA and primers

    ii) Describe what would be put into the negative well (to give a negative result) to allow comparison of A and B.

    Really appreciate your help
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    (Original post by Funky_Giraffe)


    Hey guys, really struggling with this question:

    i) Explain the effect of different temperatures (used in PCR) on the DNA and primers

    ii) Describe what would be put into the negative well (to give a negative result) to allow comparison of A and B.

    Really appreciate your help
    The different temperatures used in PCR is quite simple. The thermocycler cycles through typically 3 or 4 temperatures. It starts off at a high temperature to break the dsDNA hydrogen bonds, separating the 2 strands. It is then lowered to a temperature allowing the primers to anneal to the strands, before raising it again slightly to allow the DNA polymerase to synthesise the new daughter strands by attaching to the annealed primers.

    For the last one, to allow comparison between the wells a negative control is always used. This contains all of the PCR reaction mix (primers, dNTPs, DNA polymerase ect...) except it is missing the DNA template (in your picture's case, the animal DNA). This is so that you can confirm your PCR samples aren't contaminated (DNA would appear in the negative well if it was) and to highlight primer dimers (cases where the primer anneals to itself, which would usually appear at the end of the well in electrophoresis).

    Hope this helps
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    (Original post by Eloades11)
    The different temperatures used in PCR is quite simple. The thermocycler cycles through typically 3 or 4 temperatures. It starts off at a high temperature to break the dsDNA hydrogen bonds, separating the 2 strands. It is then lowered to a temperature allowing the primers to anneal to the strands, before raising it again slightly to allow the DNA polymerase to synthesise the new daughter strands by attaching to the annealed primers.

    For the last one, to allow comparison between the wells a negative control is always used. This contains all of the PCR reaction mix (primers, dNTPs, DNA polymerase ect...) except it is missing the DNA template (in your picture's case, the animal DNA). This is so that you can confirm your PCR samples aren't contaminated (DNA would appear in the negative well if it was) and to highlight primer dimers (cases where the primer anneals to itself, which would usually appear at the end of the well in electrophoresis).

    Hope this helps
    Thank you very much! This is so useful
 
 
 
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