Journal: Communications Biology
Article Title: Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends
Figure Lengend Snippet: T4DP treatment in the absence of dNTPs. 10 µL of H4 DNA mixture containing 50 fmol of FAM-labeled DNA was incubated without ( a – c ) or with ( d , e ) SAP for 30 min at 37 °C. Then, 0.5 units of T4DP were added. The reactions were conducted in the absence of dNTPs. After the indicated time intervals, a portion of the reaction mixture was sampled to HiDi-LIZ500 supplemented with 201 bp FAM-labeled DNAs. Data from samples taken immediately after ( a and d ), 5 min ( b and e ), and 20 min ( c ) after T4DP addition are shown. All data were y-axis scaled so that FAM peak areas of the 201 bp DNA (right most peak) are apparently even across the panels. Two calibrator peaks from the LIZ size standard (50 and 200 nucleotides) are indicated by filled triangles. The relevant size marker locations are also indicated. Peaks observed at a marker size of about 50 nt and 75 nt represent the degradation product of the FAM-labeled strand, which may be two nucleotides plus FAM (5’-FAM-AA-3’, indicated by asterisks (**)) and one nucleotide plus FAM (5’-FAM-A-3’, indicated by astersisk (*)), respectively. Electric charge of the FAM moiety might have pronounced effects on the mobility of DNA fragment as the nucleotide length decreases. The slight height increase of some peaks in ( c ) might be due to injection profile change due to prolonged incubation
Article Snippet: Fragment analysis Two microliters of GeneScan™ 500 LIZ® size standard was added to 1 mL of HiDi-formamide (Thermo Fisher Scientific, US-MA) to prepare HiDiLIZ500.
Techniques: Labeling, Incubation, Marker, Injection