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Transcript
Instructions For Use / Data Sheet IDL028 Rev Date: May 11 2015 Rev: 1 1 of 1 CG Rich Reaction Buffer (5x) Quantity IDL028 1 ml Description Templates with high-GC content are particularly difficult to amplify, due to their high melting temperatures, and may require additional measures beyond optimizing reaction conditions. Incomplete separation of DNA strands can adversely affect amplification efficiency. In addition, template secondary structure or unmelted GC-rich regions can prevent primer binding and enzymatic elongation. Co-solvents (formamide, DMSO, and glycerol) that affect DNA melting temperature are often used to amplify template with high-GC content, but reactions containing the cosolvents require significant optimization to prevent inhibition of Taq DNA polymerase. GC Rich Reaction Buffer is the co-solvent for amplification of sequences that are 50% to 90% GC without inhibiting Taq DNA polymerase activity. General Reaction Mixture (20 l) Template Primer 1 Primer 2 Taq DNA Polymerase (5U/l) 10x Reaction Buffer CG Rich Reaction Buffer (5x) dNTP Mixture (2.5mM each) Sterilized distilled water 1 ng-1 g 5-10 pmoles 5-10 pmoles 0.2-0.5 l 2 l 4 l 2 l up to 20 l Please note the above volumes are only guidelines. Optimal reaction mixture and conditions must be determined by the end user. Storage ≤ -18 oC Reference Anand, RP et al. “Overcoming natural replication barriers: differential helicase requirements”. Nucleic Acids Research. pp. 1–15. 2011 in a constant temperature freezer. Empire Genomics LLC 700 Michigan Ave Suite 200 Buffalo NY 14203 T 716.856.3873 F 716.856.3857 www.empiregenomics.com For In vitro research use only.