An Economic PCR Enhancer for GC-Rich PCR Templates
Since PCR is often problematic on GC-rich templates, we have evaluated different PCR enhancing additives and generated an Combinatorial Enhancer Solution (CES). We tested this solution on approx. 50 different primer pairs using several different polymerases
The 5x concentrated Combinatorial Enhancer Solution (CES) contains:
2.7 M betaine
6.7 mM DTT
6.7% DMSO and
55 μg/ml BSA
Add this solution to your PCR and see if it works
Some hints if your PCR still doesn''t work.
Increase the annealing-temperature in comparison to PCR without CES
Try different Mg concentrations
Check if you didn''t add to much template (more than 20 ng/PCR can already inhibit the reaction!)
Titrate the CES. In some cases, this can make a big difference
We tested this enhancer also on PCR which works fine without additive and we virtually observed no negative effects. Therefore, we are now adding this enhancer for all our routine PCR.
Notes
Betaine: We prepare a 5M stock solution, which dissolves fine when autoclaving.
Our classic PCR reactions are performed in the following buffer (final concentration) optimized for Taq DNA Polymerase (Supplementation with Pfu can increase the product quality and yield, not only enhancing the proof-reading!)
65 mM Tris–HCl
16.6 mM (NH4)2SO4,
3.1 mM MgCl2
0.01% (v/v) Tween 20
pH 8.8 (or 8.0)
and supplemented with 5-10 pico-moles of each oligo (per reaction) and 0.5 μl of 25mM dNTP mix.
If this enhancer helps you to improve your experiments and helps you to save time and money, be fair and cite the work:
M. Ralser, R. Querfurth, H.J. Warnatz, H. Lehrach, M.L. Yaspo and S. Krobitsch An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 2006 Sep 1;347(3):747-51. Epub 2006 Jul 5
-
综述
-
焦点事件
