I seem to have a huge problem. My PCR bands seems to yield the desired size however they seem to be very faint. Pls help
You need to provide more context if you want meaningful answers. That said, you can:
- Increase the number of amplification cycles
- Increase template concentration
- Try a different polymerase
- Try dNTPs of a different origin (dUTP arising from deamination of dGTP inhibits amplification by proofreading polymerases)
- Optimize dNTP and Mg2+ concentration
- Redesign the primers
Hope it helps!
You need to provide more context if you want meaningful answers. That said, you can:
- Increase the number of amplification cycles
- Increase template concentration
- Try a different polymerase
- Try dNTPs of a different origin (dUTP arising from deamination of dGTP inhibits amplification by proofreading polymerases)
- Optimize dNTP and Mg2+ concentration
- Redesign the primers
Hope it helps!
HI,
As suggested by Alejandro, you need to be specific, and his suggestion are valuable.
If you just want to increase the intensity of the bands on the gel you can use 0.5 µg/ml ethidium bromide gels. But this depends on the copies of PCR products.
I have also noticed that the template concentration in your PCR can decrease the yield in some cases based on Taq polymerase. So its better to have a DNA template at concentration of ca. 100ng. In some cases you can use DMSO to get better yields.
Hope this works,
Chetan
Hi there,
As already mentioned the context is essential to find the best solution to your problem. What is the purpose of the PCR experiment you talk about? Diagnosis? Analysis?Cloning? Screening? Do you need to quantify the product? What is the current yield of product per volume of reaction?
I wish you had provided more details and an image of your gel, anyway many factors can effect your problem Get sure about your primers whether they are well designed; Use your current PCR material except primer for different amplification reaction to find out if the problem is relating to primers. if your problem is solved then maybe the incorrect annealing temperature is the problem be sure you are using the optimized tmp you can run a gradient PCR maybe you have to decrease your annealing tmp. or maybe the primer concentration is not the ideal. maybe you will have to use a new pair of primers. if you came to this conclusion that the problem is not because of primers then you can focus on Mg concentration, your template DNA quality and possible inhibitors existence in your reaction.
also ask your friends if they are facing such a problem to check thermal cycler proficiency.
Not to sound like a broken record, but details really are needed in order to solve this problem. Previous suggestions point out a lot of important points, but in the end the cause may be as simple as using a too old stain, or with too low concentration.
When PCR products produce a single band on a gel with a weak signal, you can try reamplifying the PCR. Dilute your PCR product to 10E-3 to 10E-5 (DNase/RNase-free water is fine). Take 5 ul of each dilution (10E-3, 10E-4, and 10E-5) and use as template in a 50 ul PCR reaction using the same primers,reagents, and conditions as before. Sometimes this works, sometimes it doesn't.
I agree with Alejandro Martin. You can also optimize the annealing temperature of your PCR. If you need only a better picture, you can try to run the gel with much more PCR product.
In addition to manipulating the PCR reaction as mentioned by others. Although nowadays, its all becoming ready master mix
I use 5% DMSO (1.25 µl/25 µl reaction). This enhancer is good and makes the bands sharper and thicker, even better than betaine.
Try increasing the template concentration or the cycles ,hoping this will give sharp and good band.
Increase the number of PCR cycles
Increase template concentration
Prepare the PCR mix in ice
Optimize the concentrations of dNTPS and MgCl2
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