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I am trying to design primers using Primer-BLAST such that the forward primer spans a specific base pair site.

I am looking at KRAS for which I believe the RefSeq ID is NG_007524.1 and the forward primer should span chr12:25398285.

However, I'm not sure how to restrict the design to accomplish this. Thanks.

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  • $\begingroup$ Do you want to amplify the genomic DNA or mRNA? $\endgroup$
    – 243
    Commented Jul 21, 2015 at 0:36
  • $\begingroup$ I want to amplify DNA. $\endgroup$
    – The Nightman
    Commented Jul 21, 2015 at 0:51
  • $\begingroup$ In any case, I always verify the primers on the UCSC In-Silico PCR tool (genome.ucsc.edu/cgi-bin/hgPcr). You can never make sure enough with genomic DNA... $\endgroup$
    – WilliamL
    Commented Oct 20, 2015 at 12:54

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I think various ways can be apply, but the easiest way could be to indicate the range to set a forward primer.

Decide the length of primer you are going to get.

Let's say you would like 25 mer.

You can set the range from chr12:25398261 to chr12:25398309.

When you identify where chr12:25398285 is in NG_007524.1, you are ready to design your primers.

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  • $\begingroup$ If he had to sequence KRAS gene, he had to amplify fragments of a determined maximum lenght, depending of the platform used. Also, do not forget that many sequencers need some bases (20-40) to warm up, and some other to land. E.G. I want to sequence a fragment from bp 100 to 150 of a given gene: I will design primers starting from bp 50-70 and stop at about 170. Personally I design primers pairs for any coding exon of a gene. $\endgroup$
    – Marco Vismara
    Commented Dec 21, 2015 at 12:41

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