What is the best PCR condition (PCR mix and temperature cycles) for amplification of the matK gene with matK-S51F and matK-1210R or matK-9R primers for Myrtaceae family (genus Metrosideros)?
I have performed several optimization procedures but am still having strong primer dimer bands while the desired product is absent.
Make sure of your ordered and obtained sequences, for any mismatch made during synthesis.
And quality and quantity assessed for template must be with in acceptable range. If your template is intact, your PCR must work in general thermal and time conditions.
Annealing can be adjusted depending on the length of oligo and its Tm. extension can be depending on performance of your polymerase enzyme used in rxn.
To simplify the work you can trust using any direct PCR kit which can work with tender plant tissue like fresh tender or tough leaf or dried leaf or a part of seed or any tissue of plant, as well as with isolated DNA too. Those can perform a fast PCR and smear less gels you can see. Just need to follow its recommended steps and conditions. Example of these is Phire Plant Direct PCR (f-130) from finnzymes formerly and thermo now.
good luck!
Hey,
How did you design your primers? How long are your primers? What is the GC content or Tm of the primers? What annealing temperature(s) did you test? Are you using DNA? Do your primers span an intron (if they do, then the product will be too long for most PCR conditions)? How pure is your DNA? Do your primers bind to other regions of the genome? (you can BLAST them to find out). Did you set up a positive control (same DNA, different primers OR some DNA/primer pair that has worked previously)? Did you run a No DNA control to see what the primers do without any DNA? How much DNA did you use for each PCR? What concentration of primer did you use?
With PCR, so many things could be the issue, so without knowing the specifics of your PCR, it's difficult to figure out what it could be.
I wrote a decent description here. https://www.researchgate.net/post/While_doing_a_PCR_I_am_constantly_getting_primer_dimers_What_does_this_imply2
Best,
Casey
What can you think about?
https://ru.scribd.com/doc/226066873/DNADJ-Volume-4-Issue-1-Materialization-of-DNA-Fragment-and-Wave-Genetics-in-Theory-and-Practice
I have given Mr Turk from Illumina a thumbs up for his answer, I could not have given a better respons them him. Although Illumina is a company that performs a lot of qPCR and sequencing so they should indeed know there stuff ;)
It all starts with your primers and knowing as much from them as possible, design here can make or brake your PCR. They determine your Ta and together with the polymerase type you use the length of your PCR steps (longer amplicons need longer cycling times) and they also determine the specificity of your reaction. The less your primers can interact with each other and/ or other locations in the genome the cleaner your product and the easier it becomes to generate nice amplicons in your PCR.
Without that knowledge the rest is kind of arbitrary, you can go further but no guaranties on success and might provide a lot of optimization to get it working if it is at all possible.
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