Published: January 5th, 2021 Last Modified: January 7th, 2021
I’ve been slowly chipping away at a series of educational videos focused around DNA manipulation and cloning. I’ve taken into account the feedback from the last educational video, so things are a bit more streamlined this time!
I will be posting powerpoint slides, protocols and resources on this page as an accompaniment to the videos! Hope you enjoy! 🙂
This workshop is focused on overlap based cloning techniques with Golden Gate assembly as an interesting exception. Mutagenesis, 2 fragment and multi-fragment assemblies are covered. The intention is to equip you with a toolkit that you can apply for a wide variety of applications with a high success rate.
Every video should have the timestamps for every section in the video description. Protocols posted here will be updated as I find mistakes.
Video Links:
Powerpoint Slides:
Protocols:
Gel Purification Protocol .pdf
Gel Purification Protocol .pptx
Silica Column Cheat Sheet .pdf
Silica Column Cheat Sheet .pptx
KLD Mutagenesis Protocol .pdf
KLD Mutagenesis Protocol .pptx
Gibson Assembly Protocol .pdf
Gibson Assembly Protocol .pptx
Citations
Improved methods for site-directed mutagenesis using Gibson Assembly® Master Mix
Potapov, V., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Evans, T. C., & Lohman, G. J. S. (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology, 7(11), 2665–2674. https://doi.org/10.1021/acssynbio.8b00333
Thanks for your post! Need to take some time to finish all of these.
Very nice post! I was wondering if you have experienced problems in SDM with the KLD mix? I have tried a reaction with my favorite gene but my Sanger chromatograms are full of overlapping bases (with the wild type as the dominant peak) and they also stop after about 400 bases.
Also, some time ago I did a similar protocol before I found about your KLD recipe where I used the same enzymes but in separate reactions. Then I had the problem of having two peaks *just* at the location of my intended change. After re-transforming this plasmid, I again got the same result in some of the new colonies, wild type sequence in others. This question has also been asked before on Researchgate (https://www.researchgate.net/post/Site-directed_mutagenesis_problem_with_mutation2) but I haven’t been able to find a satisfying explanation.
Do you have any experience with this? Or a likely explanation? Thank you in advance!
In any case, big fan of your blog, keep up the good work 🙂
Hmm, interesting. So, there could be a few reasons, and how far you go depends on whether you want to try troubleshooting KLD or try an alternative method like the overlap cloning techniques. I find with KLD the reaction either works really, really well or fails mysteriously.
Overlapping peaks in a sequencing reaction could mean you have multiple templates in your sequencing reaction (multiple plasmids mixed together) or your primer is landing at multiple sites in the same plasmid. If your primer is reasonably specific, it’s entirely possible you retrieved some sort of concatemerized plasmid due to multiple copies of your PCR product blunt ligating together. You could check for this by linearizing with a restriction enzyme before sequencing, only sequence clones that look approximately the correct size. This is also assuming your minipreps are of reasonable quality, inhibitors can do all sorts of weird stuff during sequencing, worth sequencing a known good plasmid.
I think the choice of mutagenesis primer will have a significant impact on your reaction, so you should try to design a primer that has few background fragments on an agarose gel. With that said, sometimes you have little choice in the location of your primer for mutagenesis and the most you can do is play around with the lengths of the primers.
So yeah, check the size of your plasmid before sequencing with a RE, try a different sequencing primer, try a tweaked mutagenesis primer set (if possible) and always keep in mind alternative methods. Usually for mutagenesis I order both primer sets, for KLD and TEDA/Gibson as a backup. Extra 15$ for primers, but it will save you days of troubleshooting, so werf.
Thank you for the fast reply!
I designed the primers using the NEBaseChanger tool and after PCR only a band of the expected size was visible, no off-target bands could be detected. The sequencing primer was M13(-40), so I don’t expect that it would bind somewhere else on my plasmid, but you never know. The concatemerized plasmid hypothesis sounds very reasonable, I’ll check that out for future experiments. For now, I just asked Twist Bioscience to synthesize it for me, it’s a very short fragment so the price wasn’t too different from doing SDM actually.
Thanks again for your insights!