How to design qPCR primers
1. Use Primer 3 to choose a set of primers.
Go to Primer 3 (http://fokker.wi.mit.edu/primer3/input.htm) and paste the sequence of the message of interest into the input box.
Select the boxes to choose a left primer and a right primer.
Adjust the following setting leaving those not mentioned as default:
|product size range||75-200 (type in)|
|max 3’ stability||8|
|primer size||min = 19, optimum = 20, max = 21|
|primer Tm||min = 50, optimum = 54, max = 58|
|max Tm difference||2|
|max self complementarity||6|
|max 3’ complementarity||3|
Allow the program to “pick primers”.
Analyze the primer pairs to determine which is best (see mFold instructions below). If none of the primer pairs are sufficient, the make adjustments to the above settings to allow for another round of primer design (each of the options contains a link to a description of that particular variable). If no suitable primer pair can be found, the first parameter to relax should be the primer length which can be set back to the default range.
2. Check the selected primers for secondary structure formation using mFold.
DNA mFold server: http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form
Enter the sequence of a single primer. The ionic condition for Na+ is 50 mM and 3 mM for Mg++. If these primers will be used in the standard Kennedy lab qPCR protocol, the other settings can be left as they are.
Click “Fold DNA”.
Check the Thermodynamic details for each of the possible structures formed. If the Tm << 55°C, the structure should destabilize before it becomes an issue in the qPCR reaction. Anything near or above 55°C, however, is unacceptable and a new primer pair should be selected.
3. On SGD, BLAST the primers against the genome to make sure that they won’t result in additional products. For yeast, BLASTN against the REFERENCE GENOMIC SEQUENCE, change the Output Format to "Nongapped alignments" and turn Filter to "Off."
Check the sequences that the primers on an online TM calulator. If the TM is lower than that of your primers by about 10 degrees you should not have a problem with the primers binding where you don't want them to.
How to add a purchasing account and order primers from IDT
(courtesy of Monique, with minimal modification)
- Go to http://www.idtdna.com/Home/Home.aspx and set up an account.
- Continue to fill out your own user information (each of us will have our own unique username and password). The shipping address you enter should be: (Your Name), Buck Institute, 8001 Redwood Blvd, Novato, CA 94945. The billing information if requested should be Bo Khamphoumy, at the same address.
- The order link is under Products>Custom DNA Oligos. Choose the smallest (25nM) synthesis scale typically, and paste in your names and sequences.
- When you place an oligo order, select to pay by PO and use this PO number for all your oligo orders (ask someone in lab for the PO number).
- From then on when you long in with your own login and password, it will remember your shipping address, account number, and PO for you.