TITLE: Primer Design
AUTHORS: Dakota Betz, Dakota Betz

[DESCRIPTION]
Protocol for designing primers in Geneious.

[STEPS]
SECTION: Programs and Dependencies
1. Geneious
Version 11.1.5

 

SECTION: File preparation
2. You will an alignment of sequences in Geneious to design primers. Select the sequences you want to design primers for/align, then Align/Assemble > Multiple Align and then select the alignment software you prefer (usually Mafft, Geneious works too). 
 

SECTION: Primer Design
3. Select your new alignment file, then Primers > Design New Primers. 
Select Design New and check the boxes for Forward Primer and Reverse Primer. 
The "task" should remain at the default Generic. 
If you have a target region of interest, you can input that. Input your product size by checking the Product Size Between box and entering the basepair range you're aiming for (300-400 in the example below). You can also select Optimal Product Size if you would like to aim for a particular number of basepairs. 
Leave the default number of pairs to generate at 5 and Design Primers On should be set to Consensus. 
The Minimum Coverage value below Consensus Options is arbitrary--choose something that's around 1/3 to 1/2 of the number of sequences in your alignment. Click OK when done.
 

SECTION: Primer Design
4. To evaluate the success of your primer set, check the primers that Geneious has generated. If you see that they're all in roughly the same region, that region is likely conserved and will work for your target. If you see sets in different regions, take a closer look at coverage and consider ordering multiple sets. 

SECTION: Troubleshooting
5. If your primer design does not work, consider tweaking the following parameters:
Product Size Between and/or Optimal Product Size: you may need to choose a smaller number of basepairs
Minimum Coverage: you may need to reduce this number if you don't have many sequences in your alignment or if they are highly variable
Consider making a primer set for a more specific group (e.g. if you are trying to design primers at the family level, consider making multiple sets at the genus level instead). 
SECTION: Ordering Primers
6. 
 

Once on the Marketplace website select All Punchouts > IDT.
SECTION: Ordering Primers
7. Select Custom DNA Oligos and input the number of items (note that forward and reverse primers are ordered separately, so one primer set will be 2 items). Give your primers a useful name (includes what they're for, for example).
SECTION: Ordering Primers
8. Copy the primer sequences (forward and reverse) from Geneious and paste them in the sequence box.
SECTION: Ordering Primers
9. Select Standard Desalting.  Check the box to select all and then select Checkout.
SECTION: Ordering Primers
10. Check the box for a Paper Spec Sheet (this is very important--will have the info you need for programming the thermal cycler). Select your PI (Greg)  as P, then edit the product description to explain your order (designed primers, your project, etc etc).  Check that the room number and address match those for our lab at UCSD (Hubbs Hall 2350).
SECTION: Ordering Primers
11. You will have to fill out the billing information for each line item (select each one from the list at the top, then scroll down to fill out the info). Be sure to select each item and edit the Expense Type to 522401 (lab supplies). 
SECTION: PCR Program Design
12. If you're not sure how your PCR program should look, look at similar primer sets for similar genes/organisms. The following is a generic PCR program example and a good starting point. Edit the parts in bold to match your primer information (the rest can remain the same and/or be edited to your preferences):

Initial Denaturation (1 cycle)
94 °C 4 minutes
Denaturation, Annealing, Extension (30 cycles)
Denaturation: 94 °C 30-40 seconds
Annealing: Tm 40-50 seconds
Extension: 72 °C 40-60 seconds
Final Extension (1 cycle)
72 °C 5-10 minutes
Hold (temperature to maintain after program is done)
4 °C 
 



SECTION: PCR Program Design
13. If you're interested in creating a touchdown PCR modify the Denaturation, Annealing, and Extension portion of the above program as follows:
Record the final annealing temperature for your primer set (this should be  5 °C lower than the lowest Tm of your primer set).
Modify the first 10-15 cycles of DAE so that the annealing temperature begins 10-15 °C above your Tm (annealing temperature you calculated) and decreases 1 °C per cycle until you reach your final Tm
The remaining 15-20 cycles of DAE will proceed as in the above PCR program example with the final Tm. Be careful not to exceed ~35 cycles total to avoid primer dimers.