PROTOCOL 005: DESIGNING AND ORDERING PRIMERS
1. When designing primers, please observe the following rules:
Primer name: Whenever possible, primers should be named according to species of origin, gene, and location within gene. For example:
á Zf_gapdh_E1L: primer designed against zebrafish gapdh. This primer targets exon 1 from 5Õ ˆ 3Õ.
á Hs_CDKN1A_I1R: primer designed against human CDKN1A. This primer targets intron 1 from 3Õ ˆ 5Õ.
Gene name: Gene name should conform to official gene symbol, following the rules of nomenclature:
á Human genes: ALL CAPS (e.g., FOXQ1)
á Mouse genes: First letter of gene symbol is capitalized (e.g., Foxq1)
á Zebrafish genes: all lower case (e.g., foxq1b)
Primers for qPCR: Whenever possible, primers for qPCR should be designed such that:
á Amplicon size is ² 200 bp (100 – 150 bp is optimal)
á Primer pair spans an intron, preferably of length ³ 1000 bp
Primer design: All primers should be designed using Primer3 at MIT. No exceptions!
2. When ordering primers, please observe the following:
Chris Cole (firstname.lastname@example.org) will place all primer orders through IDT. In order to facilitate his job, Chris will require the following information:
á Primer name
á Primer sequence in 5Õ ˆ 3Õ orientation
á Gene symbol if applicable
á Gene accession ID, preferably a reference sequence ID
á If a primer pair is ordered, include amplicon size and whether it spans an intron (if applicable).
Zf_gapdh_E1L, 5Õ-GTGCTGCCCGCGCGATAG-3Õ, gapdh, NM_001115114
Chris will place the order and enter relevant primer information into the database.
3. When primer(s) arrives:
Chris will provide you with a unique identifier corresponding to a slot in our primer storage boxes (e.g., 15_B_2: Box 15, row B, column 2). It will be your responsibility to insure that the primer stock is stored in the correct location.
All stock primers should be dissolved in water or 1X TE to a final concentration of 100 mM.