Hello!
I am investigating plant-pollinator interactions by means of metabarcoding of pollen loads. The approach we want to use is dual-indexing in a two step PCR process. We are using four different genes (i.e. rbcLa, trnH-psbA, ITS-1 and ITS-2) and we will use Illumina MiSeq sequencing platform. In the first PCR reaction we will amplify our target regions with tagged primers (4 different tags). For this purpose, we are using modified primers which include our target region, the tags, an overhang and the illumina adapter. In the second PCR we want two uniquely tag our samples using a total of 384 tag combinations on the I7 and I5 regions.
However, and before starting with our actual samples, I am doing trials of the first PCR to see how it works. For this purpose I am using the Phire PLant DNA kit which allows you to go straight to amplification avoiding "isolation". Thus, I also prepare some trial samples which I have treated as the manufacturer of the kit says and I have used a very short cycling protocol.(initial: 5 min at 98 ºC, 20 x(98 ºC for 40s, Ta= different for each target region, and 72 ºC for 40 s), and 72 ºC for 5 min. Also I have used for each target regions different Ta. For all of them I used 55 ºC as I used this Ta when amplifying genomic DNA to build a reference sequence library of my target plant species. This temperature worked perfectly. The other Ta is calculated with a Ta calculator and based on the Tm of the primers.
As normal, I have ran the PCR and I have checked the results on a 1.5 % agarose electrophoresis gel. However, after developing the gel the results were a bit weird. I obtained bands for all my samples but these were below the the 100 bp band marked with the generuler. I do not know if this an artifact of the electrophoresis or is that my primers (60 bo kong) have dimerized. Hence, I have those bands of really small fragment size See the attached picture to know what I am talking about.
Thanks in advance
The easiest way to check for primer-dimers is to compare your reactions to your negative control (water instead of DNA or RNA). Primer dimers will still form in the negative control. Some primer sets are more likely to form dimers than others.
It looks to me like you have primer dimers and no amplified product in any of your reactions. What do your controls look like?
Dear Guillermo Uceda Gómez
Formation of primer-dimer in PCR sample is a common event. From the picture of your agarose gel, it looks like primer-dimers have formed as a consequence of the reaction. The bands of the test samples are much lower than the lowest band of the DNA ladder i.e. 100bp, we do not use so small region for PCR amplification, but this size (~60bp) is the regular size of primer-dimer.
I would suggest you few facts to check and follow:
- Reduce the amount/concentration of your primers in the PCR mixture at least 5 times to avoid the occurrence of primer-dimer as a by-product.
- Increase the cycle of your PCR reaction. Must be after reducing the primer concentration in the reaction mixture.
- Reduce the time of the extension/elongation step of the PCR according to the size of the amplified region and the processivity of DNA polymerase.
- The PCR may require more DNA sample. Due to the lack of enough DNA in the primary reaction mixture, it can not be amplified enough to be visible.
- Adjust the annealing temperature according to the melting temperature of your primer (Tm) using temperature gradient PCR or more individual trials.
- Must check the primer sequences. Design alternative primer sets if the former rules do not work.
Best wishes for your success!
The easiest way to check for primer-dimers is to compare your reactions to your negative control (water instead of DNA or RNA). Primer dimers will still form in the negative control. Some primer sets are more likely to form dimers than others.
It looks to me like you have primer dimers and no amplified product in any of your reactions. What do your controls look like?
It looks like primer dimer to me as well. Try using a hot start protocol or a hotstart enzyme which will often minimise primer dimer and run a gradient pcr over a range of temperatures if you have a gradient machine. Use less primer in the reaction ( try 1/4 and 1/16th the amount you are using now) and be sure to include a no template control in each of your experiments as Katie A Burnette suggests. Also go back to using a reliable primer set from a colleague or from your past experiments to check that the dna samples that you are making are of good quality for pcr generally.
Small nonspecific PCR amplicons. I applied these ways to avoid primer dimer, Measure and adjust primer and template concentrations to recommended values. Use touchdown PCR (temperature gradient) to take guesswork out of annealing temperatures.
Hello,
Also, for me it looks as primer-dimers. As you do not mention which is you neghative control is difficult to give you the solution. Anyway, bot replies of Dr. Paul Rutland and Katie A Burnette very good answers and I agree with what they said.
So, do it as suggested and will get good results.
Best of luck.
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