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…

2-week Lab Course

Tue, 30 Dec 2014 00:00:00 +0000

I had no idea how much work it is to organize a practical lab course. Had I known, Pirjo would have had a much harder time to convince me to give this course for the Doctoral Programme in Biomedicine (DPBM)l . Kari had warned me… The accompanying lecture course had been running from September to November. The practical course had been offered with 16 free slots, but that was totally unrealistic given that we were confined to my 23.7 square meters of lab space. Teaching lab space is available, but without equipment and all the other infrastructure that is needed for such an undertaking. 8 people registered to the practical course and - luckily - half of those pulled out in the last moment with insufficient possibility to commit to the heavy workload that the course required. Thus we ended up with four students and three projects. Under no circumstances would we have managed with more.The idea was to offer each participant the possibility to realize his own DNA cloning and protein expression project. Something that would be relevant for his own PhD studies. For that matter, I had meetings with the three groups one month in advance to plan the cloning and to order the necessary materials. We were working in parallel on the following three projects:

Recombinant DNA Technology on Michael’s Domain

An impossible overlap-extension PCR

2-week Lab Course