Has somebody experience in blunting plasmids' sticky ends using a Phusion High Fidelity (NEB)? Suggested protocols?
I has experience blunting with klenow after PCR. After PCR reaction remove dNTP and primers using a purification PCR column (as quiagen). Then incubate the DNA with klenow for 5 min at 37 C, add dNTP and incubate for additional 15 min. Stop the reaction and use for ligation
I guest the protocol for your polymerase should be the same using the appropriate Temp
Hi, blunting with proof-reading thermostable DNA polymerases works well, just assemble regular PCR reaction and incubate at 72°C for 5 minutes (maybe more for larger amounts of DNA). It is better to purify the DNA after restriction digest. Alternatively, you could just dilute/adjust the restriction mix to get salt to 50 mM and magnesium to 1.5 - 3 mM and add some Tris pH 8.3 -8.8 at 25°C.
- Badges
- Science topic