I am trying to amplify mtDNA using a hot start PCR and am having no luck.
Long PCR Enzyme from Fermentas (Thermo Scientific) with autoextension protocol, mentioned in technical brochure.....
Good Luck!!
You should google the topic Long Range PCR. Then you can find some firms which are provide kits therefore.
Another point is that you should done a temperature gradient to find out what the best temperature is for your PCR.
Arthur, I am getting no products. Tried gradient PCR. Using Elongase (OK for up to 30kB) from Invitrogen. I have designed about 10 primer pairs and tried 8 pairs with different conditions. I use a Qiagen kit to isolate DNA. Perhaps I will try another enzyme.
Dear Nafisa
I suggest you to use the mixture of enzymes, Taq DNA Polymerase and a thermostable high fidelity DNA polymerase with proofreading activity.
It is said that by this mixture of enzymes you will achieve high fidelity of DNA.
the below link may help you.
http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/long-pcr-enzyme-mix/
Hi Nafisa
All good answers above. You might want to try 5-10% DMSO. Great for amplifying long DNA stretches particularly if they have a high GC content. Also, proof reading enzymes are not as efficient as a general taq enzyme however proof reading enzymes are more specific but less sensitive. Therefore you could try a mg2+ gradient (0-2 mmol/L) coupled with DMSO.
Good luck
Cheers
Steve
Use the search function on RG and you will find several related questions and suggestions how to solve your problem.
e.g.
https://www.researchgate.net/post/Which_polymerase_do_you_recommend_for_long_range_PCR_products_20kb
https://www.researchgate.net/post/Does_anybody_know_a_good_enzyme_for_high-fidelity_long-range_PCR
https://www.researchgate.net/post/What_is_your_advise_for_optimal_long_range_PCR
https://www.researchgate.net/post/Long_Range_PCR_30_kb_Is_it_possible_from_human_DNA1
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