How do you troubleshoot non-specific amplification in PCR?
How do you troubleshoot non-specific amplification in PCR?
Use fewer cycles when template concentration is high, and use more cycles when template concentration is low. 2. Extension time was too long: Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1 min/kb.
How do I get rid of non-specific PCR bands?
You can use DMSO (0.5ul/25 ul rxn) to reduce/ eleminate nonspecific band. But when you are using it, you should increase enzyme amount by P. Also you can try using BSA. From 10mg/ml stock, you can put 1-2 ul to 25ul rxn.
What are the common troubleshooting that can occur in PCR?
PCR troubleshooting guide
| Problem | Causes |
|---|---|
| Low or no PCR product yield | Thermocycler program annealing and extension temperatures are not optimal |
| Reaction is missing Taq polymerase or other reaction component | |
| Primer concentration too low | |
| Target sequence is not in DNA template |
Which reasons often cause nonspecific bands in the PCR electrophoresis gel?
Troubleshooting PCR and RT-PCR Amplification
| Problem | Possible cause |
|---|---|
| Extra, nonspecific bands on gel | DNA contamination was introduced in primers or buffers |
| For RNA: Template is contaminated with DNA | |
| Too much template was added | |
| Primer concentration was too high |
What are non-specific bands in PCR?
Artifact or non-specific bands are bands that do not correlate to the expected mutant, transgene, or wild type bands. They are the results of primers annealing non-specifically. The presence of such bands can be disconcerting.
How do you troubleshoot multiple bands in PCR?
Popular Answers (1)
- do the reaction with a negative control (no template).
- Increase the annealing temperature.
- Redesign the primers and make the 3′ longer.
- Increase annealing time if the non-specific products are shorter than your target.
- Use less DNA template.
- Try touch-down PCR.
What are nonspecific bands?
Artifact or Nonspecific Bands: Artifact or non-specific bands are bands that do not correlate to the expected mutant, transgene, or wild type bands. They are the results of primers annealing non-specifically. The presence of such bands can be disconcerting.
What causes non-specific binding in PCR?
Non-specific binding of primers frequently occurs and may occur for several reasons. These include repeat sequences in the DNA template, non-specific binding between primer and template, high or low G-C content in the template, or incomplete primer binding, leaving the 5′ end of the primer unattached to the template.
What is non-specific binding in PCR?
Why there could be several non-specific PCR bands?
The non-specific bands could be from contamination of one of your stocks with foreign DNA (probably yours!). If this is a problem, use new stocks, always use autoclaved PCR vials and wear gloves and a lab coat. 2. Increase the annealing temperature.
What is non-specific bands in PCR?
What is non-specific binding?
Nonspecific binding is binding of the assay antibodies which is not correlated with the specificity of the antibodies. Also analytes can bind non-specifically. There are two kinds of nonspecific binding which normally occurs in the lab and which can not be distinguished from each other easily.
What to do if I get non-specific bands in PCR?
If you exhausted all the ways and still get non-specific bands. You can try purifying your target gene through gel extraction then run PCR again using your purified gene as your template. Was facing the same problem. Double check your primer sequence and try online PCR tool first.
How to reduce the extension time for non-specific bands?
Reduce MgCl2 concentration and primers also. Set gradient PCR increasing annealing temperature. If your non-specific band is at 800bp and your band of interest is at 400bp, why not just decrease the extension time? Typically protocols call for 1kb for 1 min. So for a 400 bp product decrease extension to 30 sec.
How to reduce the non-specific generation of PCR?
PCR additives, including formamide, DMSO, glycerine, betaine and PCR Enhancer (E050, E051, E055) can increase amplification. PCR Enhancer has other advantages, too, when DNA polymerase is used together, with very little magnesium ion optimization. Therefore, when we add the right amount of PCR additives, we can reduce the non-specific generation.
How can I get rid of unspecific bands in a solution?
Try to increase 3 or 4 degrees the annealing temperature. Low annealing temperatureS use to be the main cause of unspecific bands. If you have no success with this, then I would try modifying the Mg concentration or adding DMSO to the cocktail. But I think it will be enough increasing temperatures.
