A primer is labelled with dye; for example FAM, VIC, NED etc for fragment analysis using a sequencer. We see that different primers are labelled with different dyes. Is there any basis to choose a particular dye for a primer? In other words, can we choose only one dye (example FAM ) for all primers to be analyzed in sequencer?
I think that Fam and Tet give the strongest signal and are both more stable to photo decomposition than Hex. You can use only one dye so long as you separate effectively on the basis of different size if you are multiplexing your samples and any dye will do if you are analysing pcr product since you will be diluting your samples to bring them into the measurement range of your sequencer probably somewhere between 1:10 and 1:50
Just as an afterthought if you own the sequencer that you are running the samples on collect and analyse in all colours even though you only have one colour ( and the size standard which is red). This is because sometimes you get a too strong signal intensity and lose resolution but the weaker signal in the wrong colours will often show the true shape of repeat sequences better. Naturally you must also check that the correct filter set is used for your dye and that you do not use red for any samples because that is reserved for size standards
Hi Paul, we do not have sequencer but we avail the services of the sequncing company such as macrogen. And in my current study, I have labelled all 20 primers with FAM. I will be refering the PCR products to the company for fragment analysis. In this case, should I give any specific instructions as to having to the compay or can this be analyzed as usual with mix of dyes.
Hi Jigme,
That's good just tell them you want the analysis for all colours ( which I am sure they always do routinely) unless they charge extra for this. If they charge extra then add a dilution of each mixture to make sure that at least one measurement is in the good reading range of the instrument. I am assuming here that you are paying for a plate of samples not for each sample separately. If you are paying by the plate and you have some spare wells just mix samples for these wells and the mixing will cause a dilution of each sample , Phone their tech help to check but I think that they will always send an analysis on all colours
bw
paul
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology