I accidentally resuspended my stock primers (100 uM) in tris-Cl (pH 8.0) 1 M instead of 10 mM. I do PCR with 20 uM dilution from the stock. Will the high concentration of Tris-Cl inhibit PCR? I tested 3 samples and 1 amplified well, but I'm not sure if the other 2 failures could be due to Tris.
Taq polymerase works best at alkaline pH and the primer volume is a small part of the pcr rection mix so as long as you have not added EDTA to the tris I would expect the pcr to work although slightly less efficiently than in the more alkaline taq buffer. You could try the amplification again with normal amounts of primer and 1/4 the amount of primer which will still be a huge excess and see if the dilution makes any difference
Taq polymerase works best at alkaline pH and the primer volume is a small part of the pcr rection mix so as long as you have not added EDTA to the tris I would expect the pcr to work although slightly less efficiently than in the more alkaline taq buffer. You could try the amplification again with normal amounts of primer and 1/4 the amount of primer which will still be a huge excess and see if the dilution makes any difference
Monovalent and divalent salts like Na+ and Mg2+ stabilise duplex formation by shielding the negatively charged DNA sequence backbone. Increasing the amounts of salt will make more stable duplices so allowing the oligos to bind at lower temperatures. You can see this effect in washing southern blots where high sa;t is alow stringency wash and the probe anneals all over the place but low salt is a high stringency wash and much of the probe can wash off even the specific binding regions of the samples on the filter Harald Kühnel
Harald Kühnel
The figure in the link you posted is showing 'calculation of Tm from a specific primer'. Salt in purchased primers can affect your later experiments (see link below), especially for those that are very sensitive to salt. That goes for Tm too.
When, you order primers/oligos, most of the time, the company will ask you if you want to de-salt your primers (or probably what kind of de-salt procedure you want; see link below). Some experiments need extreme pure primers. However, primers from some companies always go through a basic de-salting process, which are good for PCR and some other applications (see attachment for an example).
So, when you do Tm calculation with a online platform, you can input whatever the salt concentration (or mM) is in the 'blank'. But, I would input 0 mM (since most primers ordered go through 'basic' desalting' these days). Double check your primer info sheet.
https://bitesizebio.com/36617/oligo-purification-methods/ ( Oligo Purification Methods: How, Why and for What? ) (2017)
Jo Bui
PCR buffer contains Tris-HCl. See below description from ThermoFisher. The suggestion from Paul Rutland to dilute your primers solution should be a good way to go. See attachments (click on the attachment to read).
PCR Buffer
PCR is carried out in a buffer that provides a suitable chemical environment for activity of DNA polymerase. The buffer pH is usually between 8.0 and 9.5 and is often stabilized by Tris-HCl.
For Taq DNA polymerase, another common component in the buffer is potassium ion (K+) from KCl, which promotes primer annealing. At times, ammonium sulfate (NH4)2SO4 may replace KCl in the buffer. The ammonium ion (NH4+) has a destabilizing effect, especially on weak hydrogen bonds between mismatched primer-template base-pairing, thereby enhancing specificity. Note that DNA polymerases often come with PCR buffers that have been optimized for robust enzyme activity; therefore, it is recommended to use the provided buffer to achieve optimal PCR results.
Paul Rutland and Amy Johnson
I usually add water in the purchased primers. It seems ok to me. What do you add in purchased primers to dissolve it, TE buffer? Just wandering.
Yuan-Yeu Yau I always dissolve my oligos in TE. Single stranded dna can be degraded by depurination which is pH related and the alkaline pH of TE is good for oligos. ( the water in my lab is usually slightly acid due to CO2 dissolving from the air and is about pH5.5) The EDTA chelates Mg ions and other divalent ions which are necessary for nucleases to break down DNA so no Mg means no primer degradation. Actually oligos are very stable in strong solution and if your water is very pure then nucleases are not much of a problem and many oligo making companies will recommend dissolving in water but unless tris interferes with any downstream activity I always thought it a little safer to use TE especially for longer oligos
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