Well, here's a different opinion: I usually design sequencing primers of about 20 base pairs, and try to get a 50 to 60 percent GC content. Avoid repeat areas like "AGCAGCAGCAGC" and similar. Sanger sequencing usually involves an annealing temperature of 50 degrees C, so the Tm of your seq primers should be a bit higher.
It is true, however, that using longer primers for sequencing helps you get reads that begin closer to the primer. You may have noticed that your chromatogram begins with some poor quality jumbles of colored peaks that you have to edit off. With longer primers, even the shortest fragments generated in the PCR rxn are longer, so they are not as prone to getting lost in the ethanol precipitation step that precedes electrophoresis.(That is part of the reason the beginning of chromatograms are so poor) But if you have a lot of overlap between your chromatograms, you usually don't have to worry about reading close to the primer. And yes, you can often sequence with a single primer originally designed for PCR. If you are not too familiar with how Sanger sequencing works there are some decent videos on YouTube.
Check your Gateway manual; it will explain how the recombination sites work and how you get a hybrid recombination site when it is done. The resulting att site is a little different than either of the two sites you started with. The manual should show the original sequences and the resulting hybrid. Manuals are great educators! If you don't have a paper copy, you can find it on-line.
The only difference between PCR primers and sequencing primers is that for PCR, you are often adding "tails" at the 5 prime end: tags, restriction sites and whatnot, so they wind up being quite long, sometimes. Also you have to take into account, in designing them in pairs, that they don't stick to each other to make the dreaded "primer dimer."
As for qPCR, primer design can be a bit trickier, depending on your template and purpose. Not so much the length or nucleotide composition of the oligo itself, but where in the target sequence you put them. And what protocol you are using for detection: Taq Man or Sybr Green are the most common methods. It is too complicated to get into now, but if you ever need to do qPCR you can ask for help here with your specific project. Or read manuals that come with the kits- often they help a lot.
Edited by OldCloner, 12 September 2019 - 04:45 PM.