So, I'm going to do a step-by-step about what I'm struggling with, with directions of what I'm doing.

1) Go to NCBI, select nucleotide, select cx3cl1, select mus musculus, select pick primers.

2) Change melting temp to 59, optimal to 63, and max to 65.

3) Change intron length to 200 (min) and max stays the same at 10000

4) Primer must span exon-exon junction, and intron inclusion must be checked.

5) GC clamp is changed to two.

NOW, when I get primers are larger than what they should be....

Another thing I've done when I've tried finding other primers from the same strain (not cx3cl1), I've tried different exons...but NCBI has them listed as 1-155 (this is my forward primer) and 156-276 (my reverse primer)...when I do this and keep all of the settings I geeeeeet: "No primers satisfying exon/intron selection parameters found"...

What do I do? I've been struggling with this for a month.

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