Product No. 11004760001
Random Primed Labeling probes are not recommended for detection of DNA-binding proteins via gel shift:
DIG-labeled DNA probes produced by random primed labeling yield DNA with incorporated DIG-dUTP every 20-25th nucleotide. This can interfere with protein-DNA binding when the recognition sequence is altered. For this reason, Roche recommends using DNA probes generated by 3 ' end labelling, e.g., DIG labeled oligonucleotides for these applications.
A detailed protocol is supplied in the Roche DIG Gel Shift Kit, 2nd generation, Cat. 03353591910.
High background can be due to inefficient labeling. Random primed labeling is very reliable. Care should be taken to ensure kit reagents are not degraded. Klenow polymerase is heat labile and should be stored at -20 °C. Keep Klenow on ice or in a portable cold unit.
DNA template purity is important for the amount of labeled DNA generated. Linear template DNA preparations should be as free of contaminants as possible: dNTPs in the triphosphate mixture are susceptible to dephosphorylation by kinase activity; random primers are susceptible to nucleases; and Klenow polymerase can be inhibited by a variety of protein contaminants.
Complete DNA template denaturation is also critical. Heat the template to +95 °C in a dilute buffer for at least 5 minutes. Add the remaining reagents immediately to avoid renaturation during cooling of the template.
Reduced labeling efficiency due to template contamination can sometimes be overcome by adding additional Klenow polymerase (increase to 2 - 4 μL, 20 - 40 units), and/or increasing the incubation time.