9.3 Amplification of GC-rich targets

With the TripleMaster PCR System, DNA sequences with a biased nucleotide composition (GC-content >70 %; homopolymeric stretches), repeats and inverted repeats can be amplified.

General hints

The reaction parameters, especially for GC-rich targets, may require some modification:
- Test which reaction buffer performs best with the appropriate target (Tuning Buffer versus HighFidelity Buffer) - Increase the temperature for template denaturation to 98C (not feasible for long targets >5 kb). - Use the upper limit of the time range given in Table 7 (processivity and extension rate of polymerases on difficult templates is slower). - Work at higher dNTP concentrations (300-500 M) than given in the protocol for high fidelity PCR. - For extremely GC-rich targets (>70 %) use GC-destabilizing co-solvents in combination with the Tuning or HighFidelity Buffer (DMSO: 2-8 %; glycerol: 2-5 %; betaine: 0.5- 2.5 M; trimethylammonium hydrochloride: 50-100 M; acetamide: 1-5 %). Reduce annealing temperature by 2C decrements with co-solvents. - Increase the denaturation temperature gradually up to 98C using the gradient function of the Mastercycler gradient, but restrict the denaturation time in each cycle to maximal 10 seconds. - Introduce a special pre-amplification denaturation step at 98C with the buffer/template/
'"/>

Source:


Page: All 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Related biology technology :

1. TripleMaster PCR System
2. The TripleMaster PCR System for three different amplifications
3. TripleMaster PCR System: Direct Comparison with Competitors
4. TripleMaster and Perfect gDNA Blood Mini Kit Team Up to Amplify Long gDNA Templates
5. TripleMaster PCR SystemThe Ideal Choice for Long-Range PCR
6. Using the TripleMaster PCR System for Robust Amplification of GC-Rich DNA Templates
7. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
8. Assess the In Vivo Activation of Signal Transduction Pathways with PathDetect Reporting Systems
9. Generate Adenovirus Vectors in E. coli by Homologous Recombination with the AdEasy Adenoviral Vector System
10. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
11. Enhanced PCR Cloning System