( primer mix containing DMSO (see step ...)TripleMaster PCR System

primer mix containing DMSO (see step 4 in protocol below and table 8). Protocol for GC-rich targets

1. Thaw the reagents listed in Table 8 carefully and keep on ice.
2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
3. Set up Mastermix 1 (see Table 8) containing buffer, primer, template DNA and DMSO (optional) in a sterile microcentrifuge tube, mix well, centrifuge and heat for 30 seconds at 98C.
4. Quickly spin down (2 sec) Mastermix 1 and immediately place on ice.
5. Prepare Mastermix 2 (see Table 8) in a separate sterile microcentrifuge tube, mix well, centrifuge and place on ice.
6. Just before cycling, pipette Mastermix 1 and then Mastermix 2 into a separate PCR tube on ice. Mix quickly but thoroughly, (vortex) and centrifuge.
7. Place immediately into a thermal cycler that is pre-heated at 98C (use the "Hold" step for this) and start cycling.
8. Analyze amplification products on a 0.6-1.5 % agarose gel using appropriate DNA markers.

Table 8: Mastermix conditions for GC-rich PCR ¹ increase final buffer conc. (1.6x) for targets < 500 bp or when using a low concentration of template DNA per reaction (< 10 ng genomic DNA or < 0.5 ng plasmid DNA)
² increase dNTP concentration for GC-rich targets

Table 9: Cycling program for GC-rich targets ¹ Difficult targets require more cycles (and template DNA) for efficient amplificati
'"/>

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