New England Biolabs, E5200S, LongAmp® Taq PCR Kit

Exceptional performance with long amplicons Related Categories LongAmp® DNA Polymerases Applications Long Range PCR,, Specialty PCR,, Routine PCR, FAQ Q: What is the recommended enzyme amount when using LongAmp™ Taq DNA Polymerase? A: In general, we recommend 5 units of LongAmp Taq DNA Polymerase in a 50 µl PCR reaction. For amplicons < 8 kb, we recommend 1–2.5 units per 50 µl PCR reaction for higher fidelity. Q: Can the extension step be carried out at 72°C when using LongAmp™ Taq DNA Polymerase? A: Yes, LongAmp Taq DNA Polymerase can be used at 72°C. However, extension at 65–68°C is a better choice for most amplicons. Q: What is the extension rate when using LongAmp™ Taq DNA Polymerase? A: We recommend 50 seconds per kb for maximum yields. Extension rates such as 30 seconds per kb can be used for targets up to 4 kb using a 3-step PCR protocol. Shorter extension rates such as 15 seconds per kb can be used for targets up to 2 kb using a 3-step PCR protocol on a fast PCR machine. Q: Why is the product a smear when visualized on an agarose gel? A: When PCR conditions are not optimal, a smear or high level of background is often observed. Try one or more of the following suggestions: use lower amount of enzyme use 65°C for extension raise annealing temperature try 2-step cycling protocols Q: Can the LongAmp™ kit be used to amplify GC-rich amplicons? A: Yes. The addition of DMSO up to 10% helps amplify GC-rich amplicons. Q: What type of DNA ends result from a primer extension reaction or a PCR using LongAmp™ Taq DNA Polymerase? A: The majority of the PCR products generated using LongAmp Taq DNA Polymerase contain dA overhangs at the 3´–end; therefore the PCR products can be ligated to dT/dU-overhang vectors. Q: What is the fidelity of the LongAmp™ Taq DNA Polymerase compared to Taq DNA Polymerase? A: The LongAmp Taq DNA Polymerase offers two fold higher fidelity than Taq DNA Polymerase. Q: Can NEBuilder® HiFi DNA Assembly master mix remove both 3′ and 5′ end mismatches? A: Yes, NEB HiFi can remove these mismatches up to 10bp from the end, allowing for the DNA ends to anneal without the overhang