The primers that I designed initially had Tm values of 54.26°C (Fwd primer) and 45.53°C (Rev primer) before I added the restriction sites. After I did that, however, I managed to get the Tm adjusted to 68.59°C and 69.54°C for both the forward and reverse primers respectively. This was done using the Primer-Blast tool. I do realize that the difference in the Tm value is quite large, but I'm not sure whether this will severely affect the PCR.
Would I have to set a lower annealing temperature for a few cycles first before increasing it to the Ta for my complete primers (including the restriction sites)?
Rather than alter the PCR parameters, I would see if the reverse primer can be redesigned to make its melting temperature closer to the forward primer's, perhaps by lengthening the primer sequence. It's easiest to start with a good set of primers and far harder to try and circumvent poor primer design with different PCR parameters.
Rather than alter the PCR parameters, I would see if the reverse primer can be redesigned to make its melting temperature closer to the forward primer's, perhaps by lengthening the primer sequence. It's easiest to start with a good set of primers and far harder to try and circumvent poor primer design with different PCR parameters.
If you cant redesign the primers, that sounds like a plan. Start e.g. with three cycles at 48 degrees, then three at 52 then up to 60-70. Trial and error I guess, but with a good enzyme (eg. Phusion) I think the chances are good. Also depends on template. If template is plasmid or bacterial DNA its a lot simpler than if its a large genome with higher potential for competitive binding sites...
Good luck!
Avin, if necessary, try and locate protocols for "Step-Up PCR". I recall seeing a paper about this method before but I can't seem to find it at the moment.
http://www.basic.northwestern.edu/biotools/oligocalc.html
I use this website to calculate Tm (only see Salt Adjusted that one = 58-60oC), then add your restriction cut site on the primer (but don't count it in for Tm). I use Q5 (NEB) for PCR and I manage to do around 30 plasmids so far. Hope this help.
I try to design my primers such that their Tm (without the tails carrying the restriction sites) is about the same, using the Thermofisher web site http://www.thermoscientificbio.com/webtools/tmc/ for the calculation. I use that Tm to set the hybridization temperature throughout the amplification sites. It works fine for prokaryotic DNA. Eukaryotic DNA may require more sophisticated programs, e.g. step-up), as suggested by Vegard and Mark. At any rate, if you use your current primers, you should start at least the first step of amplification based on the primer having the lower Tm without the restriction sites.
I think that design a new primer will be the best choice. You can find the another second primer, which will has a better Tm. You can create a new primer close to the previous one (only a few nucleotides) it can help.
https://tools.lifetechnologies.com/content/sfs/manuals/cms_042832.pdf
Touchdown PCR method
I use this method where better primer match is not possible. it starts with your higher pmer TM and sequentially drops per cylce to your lower TM and then has a second cycle step at standard annealing temperatures( All chosen to suit your primer conditions of course). Not sure if all thermal cyclers will do this but applied biosystems cyclers will provide this facility. i assume it will be similar to Step up but in opposite direction.It has never failed yet.
Do the restriction sites you added actually bind to template? If not you have to omitt the sequence of the RE site from the Tm calculation, so your original temperatures are the correct ones. 45 degrees is on the low side though...
The Tm is based only on the sequence that will bind to your template. Therefore, you should omit the restriction sites (and other parts of your primer that won't bind the template) when calculating the Tm of your primers. Then, the presence or absence of restriction sites in your primers shouldn't make a difference.
I use this we OligoAnalyzer online tool from IDT to calculate the Tm of my primers, it's been working very well for me: https://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
Lengthening the complementary region of your lower Tm primer should bring it up closer to the same Tm as the other primer.
I believe you primers after add-in RE sites were just much better than before. You really don't want to increase the Tm difference which will make your annealing T uneven. At your current add-in RE-site primer situation, You should have no problem to run your PCR at 55C annealing T and if you got high background then to increase your annealing T to 60 may will be fine that still have ~8C lower than your primers' Tm.
But you may need to pay attention if your over hang RE sites have extra at least 6 bp otherwise you may not cut your PCR products efficiently, which is the length RE needs to bind effectively.
Hopefully you were not waste your time and money in "redesign" your primers due to that "perfect Tm", which you don't want them have "big difference.
Good Luck!
It will not affect much. Better go with your actual primer annealing temperature as that portion of the primer binds to the target in initial steps of PCR.
In order to increase the efficiency of amplification, I think you need to keep lower Ta in the first 5 cycles and increase the Ta for the rest of the cycles.
However, You should try to amplify with a single Ta (from Tm of primers without RS) and see what is happening. If you are able to get the desired amplification with it... then you need not worry about the difference in Tm before and after adding RS to the primers.
I will agree with above scientists suggestions. higher annealing is better so don't worry, try pcr and do some changes in annealing temp. if required for better results. also if required try different primer conc.
Regards,
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