An error message may appear indicating that the PCR and primer dimer can be repaired. There are several ways to solve this problem which we are going to discuss now. We increase the annealing temperature.Increase the time/temperature associated with model denaturation.Lower primer concentration (10 pmol is definitely good)In these, use a PCR activator such as DMSO.Look at your model.Use a reinforced quality label.
Cycle time and temperature reasons
Too few points used
Using too few PCR motors may result in insufficient amplification. Use 20-35 cycles. Use less when the concentration of subroutine patterns is high, and get more value when the concentration of loop patterns is considered low.
The renewal period was too short
If the expansion time is typically too short, the target will not have enough time to replicate effectively. In general, you want the extension time to a specific minute/kb.
Vacation time was too short
If the glow time of a person is too short, the primers will not have time to attach to the device. Use an effective glow time of at least 24 seconds.
The annealing temperature was too high
If our annealing temperature is high, primers unable to bind to the structure may also be found. Typically, the annealing temperature is determined byBut 5°C lower than the Tm of the primer. Use the tool at www.basic.northwestern.edu/biotools/oligocalc to calculate the primer which is Tm). Lowest Tm for beginners when calculating the glow environment. Optimize this annealing temperature for improved accuracy with thermal slope. If the m remove primer temperature of 5°C is close to the extension temperature (72°C), consider running a two step PCR protocol. The annealing temperature should not exceed the stretching temperature.
Denaturation conditions too low
In general, if the denaturation temperature is too low, the DNA will not be easily denatured completely and the amplification efficiency may be low. Use a 95°C denaturation heater.
The denaturation time was also long
If the denaturation time is too long, the DNA may degrade. For initial denaturation currently use 3 minutes at 95°C; for the denaturation cycle, at the beginning of use 30 seconds at 95°C.
Distortion time was already too short
If the possibility of denaturation is toom is small, the DNA cannot be completely denatured, and the amplification efficiency will be low. For the 1st denaturation, use 3 min to activate the polymerase; To distort the layout during the cycle, use 30 sec
Reasons related to PCR components
dNTP concentration gets too high
When the concentration of dNTP is definitely too high, Mg2+ depletion occurs. In the final reaction, each dNTP must be present in an amount of 250 µM.
dNTP strength was too low
Each must be present at a concentration of 200 µM in the final reaction.
PCR has a high GC content in the product (>65%)
GC-enriched PCR products are difficult to amplify. To improve reinforcement, annealing prolongs the temperature. Adjust the temperature of the glow with the new temperature gradient to get more detail. You can add DMSO or additional destabilizers of the secondary structure (no more than 10%).
Model was damaged or even damaged or contained inhibitors
Matrix, rather total, will be excised or may contain PCR inhibitors. Dilute template if inhibitors are generally suspected; Otherwise, use the new model and tighten up the cycles. Try running a control reaction using a perfectly pure plasmid with template selection to decide if any inhibitory effects are present.
Primers detected impurities
Impurities in primers inhibit PCR. Use demineralized primers or cleaning primers. You can try to dilute both primers if inhibitory effects are present, but add at least 0.02 µM of each primer.
Not enough model to react
Insufficient amplification can occur if the amount of template is initially too small. Increase the number of loopbacks in a few simple steps, or increase the number of Template.DNTP
impurities were used
Impurities above the dNTP mixture may cause incomplete or erroneous amplification or PCR inhibition. Use high quality dNTP.
Primer concentrationbut it was really high
Using an excessive concentration of primers can increase the likelihood that primers will indicate non-specific binding to undesired services in or between arrays. At the end of the reaction, use well-designed primers 5 at.2–1 µM. Also make sure the manufacturer has provided the correct quantity.
Primer focus was too low
If the concentration range is too low, annealing can often be inefficient. At the end of the reaction, use well-designed primers three at a concentration of 0.2–1 µM. Also check if the manufacturer provided the correct detection.
Enzyme levels were too low
If the polymerase target is too low, all non-PCR products will be fully replicated. The optimal concentration of the enzyme depends on my complexity of length and size.
Primers have been incorrectly designed or synthesized by the user or manufacturer
Make sure the primers are in the correct order and subject to the patternWell. Use the federal government design program to avoid repetitive patterns, highly complementary regions, etc. Perform a BLAST search to remove primers that may amplify pseudogenes or that may prime certain undesirable regions. Use the entire tool www.basic.northwestern.edu/biotools/oligocalc with residual salt concentration html and 0.2–1 µM for beginners (depending on reaction conditions) to be able to calculate Tm. Use the lowest Tm of the primers.
The target was very long
PCR component concentrations and/or cycling conditions may not be sufficient for additional targets. Re-optimize the existing test design and/or increase the duration of the PCR steps, especially the add step.