We used Phusion MasterMix with 1M Betaine (5M solution from Sigma, B0300) for a GC rich template, both from cloning from cDNA and for subsequent modifications. DMSO didn't work at all, we just replace some of the water in the reaction with betaine but except for that, no changes were needed.
The most "popular" chemicals/reagents that are used for GC rich PCR amplifications are Betaine (final concentrations from 0.5-2.5M), DMSO (1-5%), Ammonium saturated buffer (concentration varies). Depending on the DNA template, product length and type of the DNA Polymerase, they may or may not help obtaining desired results. Try different combinations. I prefer three types of DNA Polymerase when amplifying difficult templates: Vent (NEB), AccuPrime Pfx (Life Technologies) and Roche produced DNA
Polymerase from their GC Rich Kit. The overall Ta of the reaction and individual Tms of each primer can do a lot of difference in my experience too. So, if your conditions for primer design are not strict, try to use a couple of different pairs.
Good luck.
GC rich PCR fragments. You will have to use an additive. Bellow I will tell you about the ones that I used but you can apply the same reason with any other additive. You have to use different temperatures to check if it works or not for your fragment as well as different concentrations of MgCl2. I normally leave 2 degrees centigrades between temperatures if I use different PCR machines and I start with 1.5mM MgCl2 final concentration. Then I go 0.5 units up if there is no yield or 0.5 units down if there are too many bands (for the MgCl2). If I use a PCR gradient I like the range 45C to 65C. If after all the troubleshooting you still have no product I will recommend to design new primers and try again (this time you can combine the PCR primers like 1aF-1aR, 1aF-1R, 1F-1aR). For my LOXL1 exon 1 I have to divide the exon into two fragment because it was too big; after designing so many new primers, It worked with DMSO 10% (both fragments) first fragment with 1nF-1gR primers and the second fragment with 1cF-1jR primers.
I have tried:
-DMSO (Sigma) - normally has worked for me between 8% to 10% of the final volume. I used this additive with Invitrogen (Taq and plat Taq).
-Betaine - normally has worked for me between 8% to 10% of the final volume. I used this additive with Invitrogen (Taq [rec Taq in USA] and plat Taq).
-FastStart Taq DNA Polymerase (the kit includes a GC rich solution - Roche Applied Science catalogue number 04738420001) - my fragment works at 8% of the final volume.
All the additives behave differently and to obtain a PCR yield it will be the right combination of PCR primers, additive, temperature as well as MgCl2 concentration.
For GC-rich fragments and difficult templates, try using Taq DNA Polymerase with Q-solution.
http://www.qiagen.com/products/catalog/assay-technologies/end-point-pcr-and-rt-pcr-reagents/taq-dna-polymerase#orderinginformation
From website:
The Q-Solution facilitates amplification of GC-rich templates or templates with a high degree of secondary structure by modifying the melting behavior of DNA. Use of this reagent often enables or improves suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer–template system and is not toxic.
Handbook: Table 5. Reaction Composition Using Taq DNA Polymerase, Q-Solution, and 10x PCR Buffer.
http://www.qiagen.com/knowledge-and-support/resource-center/resource-download.aspx?id=c73208eb-a83e-40c4-a9b6-ea5c4c94b9f4&lang=en
We also achieve far better results for this kinds of templates by replacing dGTP with 7-Deaza-2'-dGTP
You can obtain 7-Deaza-2'-dGTP from several vendor. i.e.
http://www.roche-applied-science.com/shop/custom-biotech/products/7-deaza-2-dgtp#tab-2
Good luck!
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