Usually you need to optimize for your fusion polymerase since buffers differ (or at least companies will not tell you the exact composition :) ). I guess you did not want to hear this answer. To amplify GC-rich genes you can always add DMSO up to 10% with any polymerase.  Phusion polymerase companies usually provide you with buffers for GC rich PCR which already contain secret ingredients (presumably DMSO).

To keep your cloning as simple as possible (and to make sequencing more fun) use a polymerase with a low error rate. Something which is a bit cheaper than usual stuff is Herculase (sorry for sounding like a sales person - I am not but working in the academic research as well :) )

Good luck with your amplification.

• I agree completely with Therera
• I will also add that for amplifying and cloning large and/or GC rich fragments phusion is a much better choice than Standard Taq as unlike the latter it represents the apex of new generation high processive polymerases, designed to amplify large fragments with higher efficiency than Taq; Designed to navigate regions with secondary structure, rich in GC and is also proof reading which means it is likely to incorporate fewer mis bases (errors) than WT Taq
• Regarding additives to navigate secondary structure I also agree with Theresa that (up to) 10% DMSO can be useful
• Alternatively, if you are not already doing so commence your PCR with 1 x hot start/Denaturing cycle of 96C for 5 min and then denature for double the usual time and at 96C (instead of 94c) during your subsequent standard 2 step or 3 step PCR for 30-35 cycles
• In the event of GC content > 70% you can also use 98C for denaturation in conjunction with 5-10% DMSO. However, DMSO plus denaturation temp > 96C will be rapidly denature Taq and thus it used to be common to amplify such regions to add 2 x the standard amount of Taq to a PCR reaction to accommodate such attrition 
• Better still use Phusion which has been engineered to be more thermo stable and thus denaturation @ 98C is standard. However, I would say that doubling the denaturation time from the very brief denaturation times advocated for phusion (say up to 10 sec) can stiil provide more efficient PCRs for very GC rich target regions
• Finally, for such regions a substance called betaine can be more effective than DMSO: In fact this is the principal component of many GC rich buffers (mentioned by Teresa):
• Use at a final concentration of 1M-2M in your PCR (1/5 dilution of the standard Sigma 5M stock):
• https://www.researchgate.net/post/Which_is_better_for_GC_rich_amplification_Betaine_or_DMSO
• http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011024
• Finally, some people use combinations of DMSO (5%) and betaine (1M) to increase amplification efficiency through GC rich regions (see above and attached)
• For regions with 'local' concentrations of GC the problem here is stops caused by the polymerase halting and then detaching from the template rather than obstruction of replication caused by double standed regions linked to secondary structure
• In this case supplementing your reaction with the nucleotide analog deaza dGTP can facilitate progress through these regions as incorporation of standard dGTP is rate limiting in these scanarios
• Once again for such regions phusion is superior to standard Taq

 Thank you so much. Should I use < 800bp fragment using Taq and >800bp using Pfu?

Dear Maynak

there are no advantages to using phusion for everything other than cost. Thus, if you are not likely to use up your phusion I would use that for both

In view of cost however there is no reason you should not use Taq for your smaller fragment; and phusion would always be better for efficient production of the larger fragment; especially for GC rich fragments

Thank you so much  for your advices

Dear Mayank

Temperature of annealing (Ta) are different for Taq polymerase and Phusion polymerase. The Ta for Phusion polymerase is higher than normal taq polymerase.

If you are using Phusion polymerase from Thermoscientific company (Finzyme) just use  online Tm calculator from thermoscientific. Select Phusion polymerase and enter your primer sequence (forward and reverse primer). The online software gives you the temperature for annealing with Phusion polymerase buffer. It worked for me all the time andI use Ta suggested by the software every time for Phusion polymerase.

Please find the link below to online Tm calculator.

https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html

Even NEB has the Tm Calculator but  I never tried that one.

Hope this will be useful for you

Thank you Rajesh .