User Manual
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1. 1000 2000 sooo 7000 5000 3000 1000 1000 2000 Figure 2 Fragment analysis through 3730 xl DNA Analyzer shows an oligo dC tail was added to 5 fluorescently labeled single strand DNA oligo using DNA Homopolymeric Tailing Supermix dC Arrow Tailing reaction final product A Tweenty minutes at 37 C reaction product with a 38 nt average length of homopolymer dC tail B Forty minutes at 37 C reaction product with a 45 nt average length of homopolymer dC tail Reference 1 F J Bollum Terminal deoxynucleotidyl transferase The Enzymes the third edition Boyer P D ed 3 Academic Press New York vol 10 1974 pp 145 1771 2 G R Deng R Wu Terminal transferase Use in the tailing of DNA and for in vitro mutagenesis Meth Enzymol 100 96 116 1983 1 650 872 0245 www mclab com 12. DNA Homopolymeric Tailing SuperMix User Manual 384 Oyster Point Blvd Suite 15 South San Francisco CA 94080 Phone 1 888 MCLAB 88 Fax 1 650 872 0253 www mclab com Cat NGMA 100 NGMA 200 NGMT 100 NGMT 200 NGMC 100 NGMC 200 NGMG 100 NGMG 200 Description DNA Homopolymeric Tailing SuperMix provides qualified reagents for the addition of homopolymer tails to the 3 ends of DNA with terminal deoxynucleotidyl transferase TdT Under optimized assay condition approximately average of 30 70nt oligo dA and oligo dT or 15 45nt oligo dC and oligo dG could be added to the target substrate TdT is a template independent DNA polymerase that catalyzes the repetitive addition of deoxynucleotides to the 3 hydroxyl terminus of DNA molecules The enzyme was generated from an E coli strain that carries the cloned TdT gene from calf thymus with selected mutations Protruding recessed or blunt ended double or single stranded DNA molecules serve as a substrate for TdT The addition of dNTPs to 3 overhanging ends is more efficient than with 3 recessed or blunt ends TdT incorporates dATP and dTTP with higher efficiency than dCTP and dGTP The addition of Co2 stimulates the tailing of the 3 ends of DNA fragments even applicable for incorporating ribonucleotides and modified nucleotides e g fluorescein biotin aminoallyl labeled nucleotides and dideoxynucleotides Protocol We recommend assembling reactions on
3. ice from pre chilled components This protocol is for a reaction size of 10 uL The reaction size may be adjusted as desired 1 Set up reaction as below Amount Description Final Concentration ogee 2X SuperMix 1X X pl DNA termini 1uM X uL Nuclease free water N A 10 uL Total volume 2 Incubate at 37 C for 15 to 45 minutes depends tail length expected 3 Inactivate the TdT and stop the reaction by heating to 70 C for 10 minutes or directly add 0 5ul 0 5mM EDTA Note Input quantity of DNA substrate is critical to the tail length of final product Reaction time could be adjusted according to expected tail length Repeated freeze thaw cycles may reduce SuperMix performance or TdT enzyme activity Due to the presence of CoCl2 the tailing reaction mixture is incompatible with downstream applications It is necessary to remove CoCl2 by spin column or phenol chloroform extraction and subsequent ethanol precipitation Version 1 0 1 0000 O 20000 1 0000 2000 Figure 1 Fragment analysis through 3730 xl DNA Analyzer shows an oligo dA tail was added to 5 fluorescently labeled single strand DNA oligo using DNA Homopolymeric Tailing Supermix dA Arrow Tailing reaction final product A Fifteen minutes at 37 C reaction product with a 50 nt average length of homopolymer dA tail B Thirty minutes at 37 C reaction product with a 65 nt average length of homopolymer dA tail 3000 7000 sooo 3000 1000
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