species identification: blast

primer designing: primer3 or primer blast

Hi Rubiela,

Glad you reached out! Below I have detailed several suggestions:

1) Determine the tick species you would like to study

2) Go to NCBI> Click on Nucleotides and search your tick species in the search bar (eg: 16S Rhipicephalus sanguineus). It should give you a list of nucleotide sequences of your target species (most of the time). From there, copy the FASTA sequence into your notepad.

3) Use MEGA X to align your sequences to align your target sequences. This will help you narrow down conserved areas and can help make your primer more specific.

4) Use Primer3plus to develop your primers. It's user friendly and quite simple to follow. There are various resources that highlight primer conditions. Here is one: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Make sure the primers develop fulfil these requirements.

5) To validate these primers in silico to ensure they amplify your target tick species, run the primer sequence through BLAST. Ensure the sequences BLAST your target and not other species. At this stage there will be some trial and error till you find a primer set that's specific to your species.

6) Once you obtain a primer that is specific to your species..you're all set! Send it for production

In terms of phylogenetic trees, the most basic tree would be Maximum Likelihood tree (ML) which can be done using MEGA X. Please check with your supervisor if you need to execute a more complex tree or not. If not this would be sufficient.

So you have your primer now. What do you do?

• Research previous literature on tick species identification and adapt their PCR protocols (cite them of course). See if it can apply to your primers. Do you get a gel electrophoresis band? Yes (woohoo) no? (optimise the temperature, chemical quantity, etc): Look up PCR Primer optimisation.

I hope this helps Rubiela Rosalia

Hi Rubiela,

Blast for species identification and Primer3 Input for primer designing.