HELP!!!!!! PCR

[reshma1212]

If i want to carry out a PCR for gene TP53 and i have selected my exon but exon is small and my designed primer is outside (flanking) the exon (i.e.introns) woukd my primer locations be both side of the mutation can any one explain me my qustion and also this terms flanking in this context. please suggestions requsted i am not a biochemistry student and so i am finding it very tough to deal with this subject kindly help me … 🙁

[JackBean]

what kind of PCR are you doing? Real-time or regular? Do you want to amplify whole gene or only part of it (to see the level of expression)?

Will the flanking region be on mRNA (UTR) or will you use gDNA?

[reshma1212]

I will be working on regular PCR and amplifying only a segment of the gene and will be using gDNA….

[canalon]

Introns and exons are not an issue if you are working with gDNA.
Flanking: outside on each side of the section of interest.

[JackBean]

Why do you want to clone segment of a gene from gDNA?

[canalon]

sequencing and detection of a known mutation?

[JackBean]

I don’t see any reason, why to clone it just for sequencing…

[AKumar]

How can a primer be outside the length? It is used only to start the reaction and then it is removed. Kindly optimize the parameters again according to the PCR you are using.

[canalon]

Hey do not ask me, I was just making asuggestion, and sometimes it is less hassle doing a TA cloning than trying to clone a PCR with some annoying non specific bands…
But I have no clue what the original poster want to do.
Akumar, did you read and understand the question? And do you know that primer are part of the PCR product? they are not removed!

[Author]

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