Time to make our first concerted effort in making a small site-saturation mutagenesis library. Last time I did this in any deliberate way was with the PTEN library. We used the original Jain et al protocol for doing this with inverse PCR followed by DNA phosphorylation and ligation. One of the first Python scripts I ever really wrote (back in 2016) was to generate the list of primers we needed to order to make this library. You can find the script here.
Running it should be pretty easy: If on a mac, you literally have to open “Terminal” or an equivalent Bash terminal, go to the directory with the script and write “Python 160421_PTEN_SSM_30mer.py”.
Now, if you’ve never run a script for bioinformatic -type things before, then you may not already have biopython installed. In that case, trying to run it will likely throw an error. If that’s the case, I suggest you follow the directions here to install biopython (potentially in a virtual environment). Obviously, you aren’t going to want to make SSM primers for making a PTEN library like in the script, and will instead want to make primers for making a SSM of your gene of interest in a different plasmid (ie. with different sequences flanking your gene of interest). You can literally make those changes to the script by opening the “.py” file with a basic text editor (like TextEdit), or you can use a slightly more souped up text editor like “Sublime Text”. Or you want a slightly more complicated but perhaps better long-term experience, you can install Spyder or PyCharm and make the changes in an Integrated Development Environment (IDE). Either way, you’ll want to replace the pretty self-explanatory PTEN-library specific sequences that are currently written into the script.
Some additional considerations:
1. As I noted, this is for a ligation-based inverse PCR protocol. For all future libraries in the lab, I suggest we take a Gibson / In Vitro Assembly -compatible format, where instead of a blunt ended ligation product, the primers will create a DNA product with ~18 nucleotides of homologous sequence on the ends. Instead of writing this script myself, I’m leaving it up to future students to make that script by modifying my above script. Once we create this, I’ll perhaps post it here.
2. Once we come up with the list of primers, we can order them as machine-mixed primers from IDT (and not ThermoFisher, like our standard primer orders), since I’ve recently discovered that degenerate oligos from ThermoFisher have a pretty problematic T-bias.