How can I improve my PCR results?
How can I improve my PCR results?
GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. DMSO or another secondary structure destabilizer can be added (do not exceed 10%).
How much Genomic DNA do you need for qPCR?
For digestion, we recommend using 0.5-4ug (0.125-1ug per digestion) of genomic DNA for EpiTect Methyl qPCR Arrays and 0.25-0.5ug (0.0625-0.125ug per digestion) for single qPCR assays. However, you should not use less than 0.25ug (0.0625ug per digestion) of genomic DNA for digestion.
What are some important factors to consider when setting up your PCR reactions?
Optimizing the buffer “Important factors to consider are pH, the mass of input DNA, and how GC- or AT-rich it is. Depending on the polymerase in question, different salt concentrations may be preferred and the base buffer may need to be changed (e.g., Tris-HCl vs KCl).
What can cause PCR to fail?
Usually the first thing researchers do is blame a faulty enzyme or reagent when an experiment fails but with PCR this is actually less likely to be the cause for failure. More often deeper internal problems such as primer design, thermocycler parameters, or nonspecific binding to other template sequences are the cause.
What errors can occur during PCR?
The two sources of errors which occur during PCR amplification of DNA are (1) mistakes made by the polymerase and (2) thermal damage of the DNA in double-and single-stranded form.
Can you do qPCR on genomic DNA?
As genomic DNA contains both exons and introns you cannot look at the expression of a particular gene. However you can still use your genomic DNA in qPCR which wouldn’t give you the exact expression of the gene you are looking for.
What is the optimal primer concentration for qPCR?
When designing primers, the amplicon length should be approximately 80–250 bp. A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.
How can I design my PCR primers to avoid mismatch?
Design your primers so that the digestion site isn’t in the middle of the PCR product. If the mismatch is in the middle of the PCR product, you would expect two equally sized digestion products (that would co-migrate on a gel) instead of two different sized digestion products (which could be easier to visualize and identify).
What is the minimum amount of primers required for PCR?
Primers: Approximately.5mM. Again, it is unlikely your primer concentration is to blame for complete absence of bands. Even as little as.1mM primer will be sufficient for most PCR.
Should I Digest my PCR product for ligation into plasmid?
* If you are going to digest your PCR product for ligation into a plasmid, I recommend doing the digestion before doing gel analysis.
Why are there no bands in my PCR results?
Again, it is unlikely your primer concentration is to blame for complete absence of bands. Even as little as .1µM primer will be sufficient for most PCR. Template DNA: While theoretically only one molecule is needed for amplification, realistically for a typical 25 or 30 cycle PCR this may not be sufficient.
