Taq polymerase i used was 1u/uL, Dream taq was 5u/uL. I wanted 1.25u so i took 0.25uL of Dream taq and my pcr product showed smeared images.
Various polymersases differ in their activity and fidelity. Otherwise all are same. Make sure you use the buffer given by specified company for PCR. It should work well.
As per your smear result. It could be because of either of following:
(a) nuclease contamination
(b) annealing time and temperature was too low.
(c) too much template was added
Various polymersases differ in their activity and fidelity. Otherwise all are same. Make sure you use the buffer given by specified company for PCR. It should work well.
As per your smear result. It could be because of either of following:
(a) nuclease contamination
(b) annealing time and temperature was too low.
(c) too much template was added
Smear gel image has nothing to do with the type of Taq you use. Try to optimize PCR conditions.
Good luck.
Assuming that all the reagents you used were free of contaminations, most probably you need to increase the annealing temperature for the primers to make the reaction more specific. However, it would help if you could mention the PCR steps and upload an image for the gel run.
Also please follow this https://www.qiagen.com/de/resources/faq?id=4eb03cc8-4623-4e9e-96b2-6a4c17c03c58&lang=en
I did gradient PCR to optimize my PCR conditions, faint band was observed at the expected site at 55 0c along with many non specific bands. afterwards, on doing conventional PCR at 55 0c no bands were seen instead there was smear. i also tried 57 0c but again smear was seen.
Have you tried to lower below 55 oC? You can try a temperature gradient of 50-55 oC.
Use high fidelity polymerase enzymes to get the pcr product and reduce the annealing temperature
One thing you can also do is to check if the primers you used doesn't have repeated sequences on the plasmid or DNA template you use. Secondly if you use a lot of DNA template you might also see a smear as result. Try to lessen your DNA template the next time.
You have to include controls and look at the whole gel. Was the sizing ladder a smear as well? How about the no taq control reaction? As the others have suggested you need to titrate how much template you use, optimize and validate your reaction before starting data generation.
Chongtham Rajiv i used gradient of temperatures from 48 to 57 oC. it worked best at 55oC else at other temp no bands were seen
Carol M Bruzzone in my case controls were also not working. control samples did not show any band. ladder was not smeared it was ok and i didnt used any no taq control but i had no template control and it showed only primer dimer
my DNA conc was 87.66 ng/uL and i took 2 uL for 25 mL reaction volume. was that too much to be used for amplification ? i attach my gel picture for further conclusion to be drawn.
Try to decrease the concentration of the template around 10-20 ng per reaction. Some times the PCR inhibitor may contaminate the samples during DNA isolation procedure.
Also what sample are you using and have you tested primer specificity?
You can try NCBI primer blast to authenticate your primer.
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Good luck....
Thanx Rajiv!! I have selected the primer i am using from literature and blasted it. I am using DNA samples of Chlamydia psittaci. I got the DNA of the bacteria in FTA cards from Norway, i extracted DNA from it and did PCR.
Try this one: https://www.protean.bio/en/recombinant-protein/58/tick-taq-dna-polymerase
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