An impossible overlap-extension PCR

Sat, 21 Jun 2025 00:00:00 +0000

A PhD student of mine asked me about plasmid maps for several DNA constructs that I had created some 20 years ago. Since SnapGene did not exist at the time, I had used the now obsolete GeneConstructionKit2. I performed many clonings at the time, but I did not continue generating maps for all of them. The GCK2 format does not allow for easy annotation. You needed a separate program for comprehensively annotating the plasmids, and this data was saved in a separate file, the so-called “illustration” file: what could possibly go wrong? Yesterday, I ended up digging out my old lab notebooks and retracing about 10 old clonings in SnapGene. However, I was unable to simulate one of the assemblies because SnapGene was too conservative in disallowing “bad” PCR primers to function. I had performed overlap-extension PCR to introduce a mutation into the mouse VEGF-D cDNA. The homologous mutation had been introduced into human VEGF-D before, and I therefore had the primers for the human sequences. Mouse and human VEGF-D are very similar. The primers designed to amplify the human PCR were not perfect when using mouse cDNA as a template, but none of the differences would result in amino acid changes. So I attempted the PCR with a primer that had a mismatch in the third nucleotide from the 3’-end. The PCR was successful, but even when I lowered the hybridisation parameters to the least stringent settings, SnapGene would not anneal this primer to my template. To simulate cloning in SnapGene and generate a map, I needed to introduce a mutation into my primer and then reverse the mutation after the overlap extension PCR. I guess I need to file a bug report (or would this be a feature request?). It seems appropriate that the program should be able to allow annealing of primers that do anneal in reality…

Low-budget PCR-based mutagenesis kit

Thu, 26 Nov 2020 00:00:00 +0000

If you need to modify your DNA construct, one frequently used method is the PCR-based mutagenesis, where you incorporate the mutation into the middle of two complementary primers. Alternatively, you can use a primer tag for longer insertions. Then you simply amplify the whole construct by PCR.This works reasonable well for constructs smaller than ~10kb. There are several commercial kits available for this purpose that differ in the details. Among them are the QuickChange kit from Agilent and the Q5 SDM kit from NEB and the Phusion Site-Directed Mutagenesis kit from ThermoFisher. The QuikChange kit is the most expensive (also because you always buy it together with competent cells) and the NEB kit is rather on the budget side (168€ for me here in Finland). Most of the kits are sized for 10 reactions. But what do you do if you want to do only one or two mutagenesis reaction. Do you buy the whole kit? I faced this question two weeks back. I went to our enzyme freezer and realized that we have T4 DNA ligase, Polynucleotide kinase (PNK) and Phusion polymerase. That is all what you need to make your own site-directed mutagenesis kit!

Mutagenesis on Michael’s Domain

An impossible overlap-extension PCR

Low-budget PCR-based mutagenesis kit