This is the PCR products of genome DNA of seaweeds. Though I did the PCR twice same results were obtained. (1kb ladder has been used)
I see amplification above primer dimer, I just think the mix is very salty/dirty, may be your original genomic even
It seems there is no amplification and some primer dimer has been formed so change the primer or change the temperature and time. Try this time with the modification.
There is no amplification. I think you should revise your primers (Forward and revers ) maybe one of them is wrong if they are correct you need to optimize all chemical, time and temperature again.
Good luck.
Once you could check your primers, either it has been high content of GC or melting temperature. otherwise you can keep the gradient PCR with different temperature which is near to Tm of primers some times increase the temperature or add Mg 2+ are required in optimum concentration for the activity of most thermostable DNA polymerases as well as for several other steps in PCR. So it will be worked.
Good Luck.
Hello,
Could you please give some information on the amplicon size and cycling conditions? From the picture, it is clear that there is no amplification and you see primer dimers and smear. You could play with cycling conditions but we can help only if we know what you are doing.
This is quite a strange 1 kb DNA ladder. It has very few bands and I guess the lowermost one should be 250 bp. If so, from the figure it is obvious the amplicon size is near this size and the primer dimers should be expected at lower sizes. The smear might appear if you put a high concentration of DNA in the PCR reaction. In any way, you should provide more information like Nivedita suggests if you look for help.
Thank you all for the replies....
Then I revised the PCR with 10 times diluted DNA samples and here I have attached the picture of it. 100bp ladder has been used. Size of the amplified DNA is 1.7kb.
Hello,
I see that there is no amplification. I am worried that your cycling conditions are not appropriate. Could you provide some more information on that please? Additionally, please check the quality of your template once. Is it possible to have some controls with the PCR, negative or positive or both, it helps great deal you know.
All the best.
I agree with what people have said above, there's been no amplification of the target. Get a positive control of some sort on the gel so you can see whether the primers and reaction conditions are able to appropriately amplify the target of interest and then troubleshoot from there, only changing one thing at once until you've discovered the problem.
What percentage gel are you using by the way?
Hi,
Clearly no amplification. But in subsequent experiments, proceed with diluted DNA template. i would suggest a temperature gradient PCR. Other conditions that you are using, would be checked by using appropriate positive negative and null controls. Also, u must check for the quality of DNA.
good luck
Dear Sachithra,
I think you have already got a lot of useful advices and you have now a lot of work with it. Please also tell us how many cycles are you using. More information can help to better see in your protocol.
Try waiting until the whole content of your buffer, oligos and dNTPs tubes has melted. Include a positive control to assess if your problem is caused by the sample or the reaction (even sometimes the PCR machine block fails).
I have seen results like this before but I have not exact clue of the origin as I have changed everything at once (oligo alicuots, dNTPs, Taq, Thermocycler and also pipettes).
Good luck.
I see amplification above primer dimer, I just think the mix is very salty/dirty, may be your original genomic even
If there is suspicious that the DNA isolation solution is not clean, it pays off to go one step back and do it again.
- Badges
- Science method