Modification of DNA fragment ends prior to ligation is an essential step to the overall success of a cloning experiment. With the Thermo Scientific FastAP Alkaline Phosphatase and Thermo Scientific Fast DNA End Repair Kit, modification of DNA ends is fast and effective.
FastAP Alkaline Phosphatase is ideal for rapid and efficient dephosphorylation of linearized vector DNA in just 10 min. Complete thermal inactivation of the enzyme in just 5 minutes makes FastAP a faster and more efficient alternative to SAP.
The new Fast DNA End Repair Kit is designed for fast and efficient DNA fragment blunting and phosphorylation prior to ligation. This kit can be used with all types of double-stranded DNA:
Efficient ligation of DNA fragments following end repair with the Fast DNA End Repair kit
A 130 bp PCR product with 3'- dA overhangs was end-repaired using the Fast DNA End Repair Kit and kits from other vendors according to supplier's recommendations and subsequently ligated with T4 DNA Ligase (#EL0014). Ligation conditions: 200 ng of a 130 bp PCR product ligated with 5 U T4 DNA Ligase in 1X T4 DNA Ligase Buffer with 5% PEG 4000 for 1 h at 22°C.M – GeneRuler™ 1 kb Plus DNA Ladder (#SM1333),C1 – PCR product,C2 – PCR product after ligation, T – PCR product after end repair using Fast DNA End Repair Kit (20°C, 5 min) and ligation,A – PCR product after end repair using Vendor A kit (20°C, 30 min) and ligation,B – PCR product after end repair using Vendor B kit (RT, 45 min; 70°C, 10 min) and ligation.