PCR on Michael’s Domain

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!

Kapa HiFi excels, Phusion works sort-of, Q5 and Platinum SuperFi disappoint

Fri, 02 Dec 2016 00:00:00 +0000

My last NEBuilder assembly got stuck because I couldn’t get two of the PCR reactions to work. I needed one small fragment and two ~3-kb-fragments. I needed to insert a 2A-sequence in between the small and one of the 3-kb-fragments and thus added the necessary sequences as tails to the primers. The template was not especially GC-rich, nothing too complicated, but only the small fragment did amplify in my first attempt. When also my second attempt failed to amplify the 3-kb-fragments, I decided to try out some alternative polymerases. For cloning purposes, I have been using exclusively Phusion High Fidelity DNA polymerase (from New England Biolabs or from ThermoFisher ) for the last years, but maybe there was something better and more robust? I received three different free samples for testing: KAPA HiFi HotStart , NEB Q5® High-Fidelity and ThermoFisher Platinum SuperFi™ . My graduate student did the PCRs yesterday and I ran the gels today. From the gel quality, you can see that I don’t have much routine anymore, but the overall results are quite clear and in the future, our go-to polymerase for tricky templates will be KAPA. I think KAPA’s proofreading capabilities are a bit below Phusion, but I do not care if 2% instead of 0.5% of the DNA products contain a mutation.In the attached PDF file, you can see, that my grad student included two more samples for the Phusion polymerase, where she used the same conditions, but a different template (supercoiled plasmid instead of linear DNA). Surprisingly, the Phusion polymerase did a much better job to amplify from supercoild DNA than from (the same) linear DNA; I cannot explain that…

How much PCR product can you get?

Thu, 10 Mar 2016 00:00:00 +0000

How much PCR product does one get from a typical PCR reaction? 50-120 ng/µl seems to be a typical result, but it very much depends on your PCR conditions. If you need to minimize your primer concentration to maximize specificity, your yields can be significantly below that. Vice versa, if specificity is not an issue (e.g. for some PCR clonings), you can get many times more.Let’s consider the typical maximum amount of a single PCR reaction, which is 100 µl. And let’s assume we do not have specificity issues and therefore we can use large amounts of primer (1 µM each) and dNTPs (0.2 µM). Since the synthesis of every molecule of double-stranded PCR product consumes one primer, the theoretical maximal molar concentration of double-stranded (ds) DNA product is the same as your primer concentration: 1 µM. 1 µM dsDNA would equal 100 pmol for a 100 µl PCR reaction. How much is that in micrograms? That is of course dependent on the length of your PCR product: e.g. 100 pmol dsDNA of 1000 bp is equal to 66 µg.Can you really get that much? Not in our example of a 1000-bp-product. The reason is that the building blocks of the DNA, the dNTPs, become exhausted long before the primers do. For the 1000 bp product, only 40% of the primers are used up when the dNTPs run out (assuming a GC to AT ratio of 50:50 in your amplicon). To make one molecule of a 1000 bp dsPCR product, you need about 2000 molecules of dNTPs. For our example, a 400-bp PCR product would therefore be optimal as both primers and dNTPs get exhausted at the same rate.It seems that if you want to get larger amounts of longer PCR products you would need to increase the dNTP concentration. However, in our example of a 1000 bp product, the theoretical maximal amount of PCR product is about 26µg, which is massive and sufficient for most applications. There is an online calculator, that lets you play around with primer and dNTP concentration and product length: http://www.bioline.com/us/media/calculator/01_14.html

Formula to Calculate the Annealing Temperature of Oligonucleotides for PCR

Wed, 30 Aug 2006 00:00:00 +0000

The thumbrule for calculating the annealing temperature for a PCR primer isTm (°C) = 81.5 + 0.41(%GC) - (675/N) where %GC is the percentage of G and C nucleotides in the oligo and N is the length of the oligo given in nucleotides.