Quickly design protein fusion primers using MS excel
I’ve been doing a lot of cloning lately and wanted to share my strategy for fusing genes in a relatively quick and painless way. This strategy works well when you’re putting on the same series of fluorescent protein tags to different proteins, like Blue, green, yellow, and red, onto protein A, B, and C. This works especially well for re-tagging a receptor with a different fluorophore, although you still need to verify that your protein is functional.
1. Determine the 5’ and 3’ primers that will PCR amplify your fluorescent tags. These will always be the same, the only difference is the sequence of the gene that youre fusing the fluorophore to. These sequences will be added to the left of the fluorescent tag sequences.
2. Determine the 5’ and 3’ primers that bind to the gene you’re trying to tag.
3. Copy this data onto an excel spreadsheet
The primers that anneal to your gene of interest (5’ and 3’ respectively). PCR will add this to your fusion protein primers so that you can perform SLIC.
The 5’ and 3’ primers that anneal and amplify your protein fusion tag
4. Since you are going to add the same gene of interest sequence to the 5’ and 3’ fusion primers, just design one generic group of primers first, but don’t put any specific sequence in yet, just put something that identifies the 5’ and 3’ end. I did (f) and (r).
5. Cut and paste the info from step 4 into different excel books
6. Name the books as your gene of interest
7. Copy the gene of interest primer info into the respective excel books.
8. Use the find and replace function to add your 5’ gene of interest, meaning find “(f)” and replace with your gene of interest primer sequence.
9. Repeat step 8 for the 3’ gene of interest primer. For me it was (r).
Excel does all the replacing for you, including renaming things. Way faster than making a bunch of maps yourself, then copying and pasting primers from each map.
10. Rename the primers according to your gene
11. Copy and paste this info into your oligo synthesis company of choice
12. PCR amplify your protein tag when your primers arrive.
Since your adding sequences that aren’t there, these won’t affect the annealing temperature of the primers, so you can technically do all your different primers in one go. For example, all moxBFP tags will have a Tm of 70°C, all mCherry will have 68°C. If you’re really careful, you might even design the generic primers to have the same Tm as one another so you really only need to run one temperature.
The first time I tried to tag a group of receptors with fluorescent proteins (FPs) it was overwhelming. This simplifies things a little bit, so that you don’t have to make a bunch of plasmid maps before you start primer design. Eventually you’ll need to make a plasmid map, although even if you make the plasmid map (for me it would’ve been 16 different maps) its still going to take a long time to design and copy the 5’ and 3’ primers (32 total primers). Also, you want to double check that your primer sequences are correct, but if you copy the info from 16 different maps, your going to have to click through a lot of info. With this, you only must check the 5’ and 3’ primers at the very beginning of the design process. As long as you don’t accidentally hit a key, the primers will always be correct, since excel won’t make a mistake when it replaces the (5’)/(3’) with the gene of interest sequence.
This technique is really useful when combined with SLIC, because to tag a lot of proteins really quickly, you only have to amplify the “backbone” plasmid, which has your gene of interest, but no fluorescent protein/tag info.
