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Picking primers with ApE
There are many software tools available to select primers from a certain template (primer picking).
There is also a function in ApE for primer picking. We will use this function for picking primers for the URA3 gene.
Follow the instructions below on your own computer:
Open the URA3_locus.txt file in ApE. It contains a FASTA file or the URA3 locus of Saccharomyces cerevisiae. URA3 is an enzyme in the metabolic pathway for uracil synthesis.
The sequence should be 2804 nt and contains the gene (ORF, open reading frame, 804 nt) and 1000 nt sequence before and after the gene.
Select the first 500 nt (you can use the Edit>Select From-To.. from the Menu) or select manually. The selection will tell ApE to look for primers in this area.
Select Tools Find Primers…
You will see a window like the one above.
Change the following settings as indicated above in orange::
Length Minimum 20
Length Maximum 25
Tm Minimum 50
Tm Maximum 65
Click OK.
You will get a series of primers in a window, click on the first one to select it in the main sequence window.
The first primer will be 20 nt long, start at 64 and end at 83. What you have selected is a forward primer for the sequence. Copy the sequence for the primer to Notepad (Bloco de notas) or another text editor.
Question 1:
Calculate the SEGUID for the primer, the first five characters are geW47. What are the last five?
Now we need a reverse primer! Select the last 500 nt of the sequence (2304-2804).
Select the Find Primer function as before.
Be sure to select the same temperature (Tm) limits as before
Select the opposite Orientation this time: 3’←5’
Question 2:
Calculate the SEGUID for the primer, the first five characters are H/hwG. What are the last five?
Question 3:
Assemble the PCR product from the two primers and confirm that the size of the PCR product is 2731nt and calculate the SEGUID
The first five characters of the SEQUID is zIIiW, what are the last five?