i tried gradient PCR for a new set of primers and got the amplification. However, when i tried running the same set of primers on a single annealing temperature, it showed no or weak amplification. Kindly suggest what is the reason behind this .
As you are using the same pcr machine I think there may be some small dofference in tube temperature between the two runs. For instance if the good result is at Ta 60 in the gradient then the 2 positions either side of it may be 58c and 62c. This is different from a single block pcr where all positions are the same temperature at the same part of the cycle so I wonder if , in the gradient, the real temperature in the 60c well is a little lower than that because of the 58c position next to it so the sample reaches 60c a little quicker and remains at 60c a little longer than on a single block pcr where all temperatures are the same. If so then either set your Ta a little lower or hold at the TA for a little longer. I am assuming that all volumes are the same between the 2 runs. Often temperatures will overshoot a little and bounce back to the correct temperature on many machines and this effect could be magnified on the gradient. For instance Check the ramp time on a gradient run and adjust the ramp time on your single temperature pcr to be the same as the gradient run. If the gradient run is 3c/second make sure the normal pcr run is also 3c/second
Did you keep the same samples between these two experiments? It may only be that your gene is not or weakly expressed in these samples. If you use diluted DNA maybe you can try to keep it more concentrated
Hi Bhoomika,
If you could see amplification with gradient conditions, it has to work with single annealing temperature as well. If you are seeing weak or no amplification, it indicates that the primers are not able to bind to the template. Consider the points below while setting up a reaction next time.
1.Are you making a master mix and then distributing them to various tubes? Ensure all the contents are mixed evenly in the solution. You can achieve either by vortexing the master mix, ( for a sec or two, atleast thrice) and spinning down the sample for few sec's (very imp) before aliquoting.
2. Ensure you are adding sufficient primers and template and that they are available for the rxn to take place (short spin and good pipetting helps. Also, see if u are able to find a faint primer-dimer at the bottom of the gel-which tells you if the diluted oligos that you are using are sufficient for the rxn or not)
3. Try to increase the annealing time by few more sec's , while working with single amplification temperature.
Hope it helps and all the best !!!
Hello,
As mentioned above, sounds like your primers are not binding in the single annealing temperature program, likely meaning the annealing temperature is too high. If you haven't already I would try using the lowest temperature of the gradient for the single temperature amplification.
If you determine the optimum annealing temp for your primer sets with temp gradient, you should see same amplification for second reaction at optimum temp. You may have a pipetting problem or DNA template problem. You must try DNA template optimization at optimum temp.
I would suggest to repeat the same gradient PCR to see whether you get the same result or not... check how sharp the desired bands are.. maybe you will choose different annealing temperature depending on your gradient PCR result.
sometimes the template type you use is important as well. genomic DNA? cDNA? plasmid DNA? or,,,,? maybe the amount of template is not sufficient for PCR I mean it is less and because of improper distribution it gets more less so PCR is not reproducible! Normally one can achieve desired result easily and in a wider range of annealing temperatures if plasmid DNA is the template and on the other side cDNA can be a little bit problematic if your desired gene expression is less so mRNA amount and cDNA will be less subsequently.
as a conclusion, for me if you are sure about your annealing temperature get concentrated on your template concentration however I suppose that the other factors such as your thermal cycler, other PCR reagents in each set are ok and there is no problem with them.
Dear Bhoomika Sharma
Viability of the template a potential problem. Contamination with DNase. use fresh dNTP's and Taq, also hygiene in the work area.
Pls. find the attached files
http://www.techne-equipment.de/adminimages/T01_002A_PCR_trouble_shooting_guide.pdf
http://www.drugdiscoveryonline.com/doc/using-gradient-pcr-to-determine-the-optimum-a-0002
http://primerdigital.com/pcr.html
http://bitesizebio.com/343/the-essential-pcr-troubleshooting-checklist/
Hoping this will be helpful
Regards
Prof. Houda Kawas
Did you diluted each primer and then mixed them in your reaction? If you did that, that might be the reason. Because your primers both diluted too much. In case you want to work with many primers in multiplex you should dilute once all the primers (after mixing them). Try this !!
Many factors can influence the PCR reaction as MgCl concentration (25-50 mm) commercial kits may vary in these two presentations, another possible cause is the quality of extracted DNA can be degraded and this show weaker bands.
are you using the same pcr machine for the gradient as for the single temperature amplifications please?
As you are using the same pcr machine I think there may be some small dofference in tube temperature between the two runs. For instance if the good result is at Ta 60 in the gradient then the 2 positions either side of it may be 58c and 62c. This is different from a single block pcr where all positions are the same temperature at the same part of the cycle so I wonder if , in the gradient, the real temperature in the 60c well is a little lower than that because of the 58c position next to it so the sample reaches 60c a little quicker and remains at 60c a little longer than on a single block pcr where all temperatures are the same. If so then either set your Ta a little lower or hold at the TA for a little longer. I am assuming that all volumes are the same between the 2 runs. Often temperatures will overshoot a little and bounce back to the correct temperature on many machines and this effect could be magnified on the gradient. For instance Check the ramp time on a gradient run and adjust the ramp time on your single temperature pcr to be the same as the gradient run. If the gradient run is 3c/second make sure the normal pcr run is also 3c/second
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