How do you prepare dilutions of primers in order to send DNA into sequencing?

Question

Why do we have to add reverse and forward primers to a sample of DNA before sending it to sequencing?

What is the process to prepare the primers? (make up your own numbers and examples if it makes it eaier to explain)

Answer 1

Modern sequencing machines build copies of your template DNA using labelled nucleotides that can then be read by their fluorescence at different wavelengths of light. Just like in PCR, you want primers to start the sequence going so the copies can be built. You don't want to add both forward and reverse primers to the same sample tube, though - you'll need 2 separate tubes; one for forward and one for reverse. The primer dilutions pretty much depend on what the people running the machine want you to use; it's a function of the kind of machine and what their experience tells them works best. To make the dilutions, I usually start by taking my lyophilized stock and diluting it to 100 uM (same as for PCR primer reconstitution) and then diluting from that parent stock as desired. Since 1M = 1mol/L, then we go 100 uM = 100umol/L = 0.1umol/mL = 0.1nmol/uL. So, if you've got 75 nmol, you would dissolve that in 750 uL of water to get a 100uM solution. If you want a 1uM final primer concentration for sequencing, then you would add [1/100 of your final volume] of your parent stock - for a 50 uL sample, you would want 50/100 uL. That's not much, so I would first dilute my parent stock by 1/10 to make a 10uM working stock, and use that for my final addition - 50/10 uL is more accurately measured than 50/100 uL... Hope this helps!

Answer 2

Primer Dilutions

Answer 3

It depends on where you're sending it to sequence. If you're using standard commercial primers (e.g. M13's, T7), the sequencing facility usually does not need the primers.

If they are custom primers, then obviously they need your primers, and it makes more sense for you to send it to them. Adding the primers saves the staff at the facility time.

If you're doing in-house sequencing, then to save time and money (which is why you get the cheaper price), they usually ask you to do most of the prep.

There are instructions from the facility telling you in detail telling you what their preferred concentrations of DNA sample and primers are. Dilutions are just a lot of math, which is annoying, but it's really not that difficult. If you're having trouble, don't be afraid to call Technical Support; biotech companies' tech support are great in my experience.

If you don't like doing the work, then there should be several facilities in your area where you can just give them your DNA sequence, what primers you want with it, and the concentration. You'll likely have to pay more, or at the very least wait a little while to receive your sequence.