My template amplifies when I use Hi fidelity kapa and not when I use Taq polymerase (normal/regular) . Is there any limitation to template size for taq polymerase? I had given it 1 minute and 45 seconds for extension and it still did not amplify.
Buffer used for the polymerase also affects the annealing temperature for the primer by a few degrees. A temperature optimization might be required. If its a high GC content template you are using, try adding DMSO. For different polymerases provided by NEB you can check the annealing temp on https://www.neb.com/sitecore/content/nebinternational/home/tools-and-resources/interactive-tools/tm-calculator.
Of course yes, it should amplify 1500bp. Just double check the enzyme provider's protocol and the buffer you are using! (co factor must be there in the buffer).
First thing is to check is if your Taq works by trying to amplify a small product like GAPDH. Then if it works fine increase the MgCl2 concentration and try to play with annealing temperature. Put a gradient PCR for larger range 55-65 and see you get the product. If nothingamplifies, try addiing DMSO, betaine and glycerol. Good luck
I think you are referring to Kapa HF polymerase from KapaBiosystems. This polymerase has been engineered to have an increased affinity for DNA without the need for accessory protein domains. If you are using Taq, maybe try adding some single strand binding proteins in your reaction.
Check the template quality...shearing will lead to non amplification by Taq polymerse...
Have you run positive control with Taq polymerse to know whether its working properly or not.....
If everything is fine with your reagents then try adding 1-5% DMSO...or you can also run touch down PCR...if product is needed for cloning.....otherwise gradient PCR
Actually Kuldeep, 16S rDNA might not be the best choice for a control as commercial Taq DNA polymerase is usually contaminated with bacterial DNA from a variety of sources. In order to do bacterial identification through 16S rDNA sequencing, I had to switch to using DFS Taq.
If you can amplify from this template using a HF polymerase, I would determine the true extension time of your Taq. You can do this by amplifying a known gene of a purified plasmid template. Use progressively shorter extension times until you can't observe a strong amplicon.
In my experience Taq from different commercial sources have dramatically different extension times. Takara ExTaq can generally do 1 Kb in 30 seconds, even though they suggest 1 min (I believe?) where as Fermentas or BioShop Taq takes around 90 seconds per Kb. Personally, I don't even use those enzymes to A-tail for cloning.