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.
Try to tinker with the annealing temperatures in the program and DNA concentration. Taq polymerase can amplify 1.5kb
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).
thank you all for your suggestions. yeah the catalog says it can go up to 5kbp. I guess i ll just trying playing with Mg2+ or temperature.
Some DNA fragments cannot be amplified by Taq polymerase but only by phusion or high fidelity . I have faced a similar problem
we used taq to amplify 4.2 Kb.. it depends on company u use..try to add salts MgCl2 /MgSO4 and enhancers like DMSO4/ or glycerol.
What is your template and are you using primers that anneal perfectly?
If your template DNA is low in quality (fragmented or damaged), you may face problems amplifying the target sequence with Taq.
Mismatches between the primer and the template can also block/lower the efficiency of amplification by Taq.
The exonuclease activity of a proof-reading polymerase allows it to overcome such problems.
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 th e template for GC content. If it is high, use DMSO. That helps. Also, you should check the template quality.
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
Try to amplify 16 s rRNA gene from your template by using full length universal primer by using you taq pol, to ensure it is working.
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.
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