For evenly labeled, full-length coverage, DNA Polymerase I (E. coli) (M0209) is the preferred enzyme. At elevated reaction temperatures, Taq DNA Polymerase may also be used.
For high yield, short, fragmented (~250 bp) dsDNA generation, Klenow Fragment (3'→5' exo-) (M0212) is recommended.
Nick Translation
When using nick translation to generate probes, DNase I is typically used to nick the DNA. DNA Polymerase I (E. coli) removes leading bases at the nick with its 5'→3' exonuclease activity and then fills in with labeled bases.
Modified Nucleotides
Since incorporation efficiency of modified nucleotides is generally lower than their natural counterparts, a 100% replacement can cause the polymerase to stall. To balance yield with labeling efficiency, we generally recommend testing several ratios of modified to natural dNTP.
The following polymerases are not recommended for labeling or partial fill reactions because their 3'-5' exonuclease activity is difficult to control:
- DNA Polymerase I, Large (Klenow) Fragment (M2010)
- T4 DNA Polymerase (M0203)
- T7 DNA Polymerase (M0274)
