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7 Nov 2013 I need help with overlapping PCR, I have 4 primers A, B, C and D where B and C have complementary bases. 1. first PCR run with Phusion
 Their goal was to assemble these 3 fragments by PCR overlap extension. They tried to perform this using Phusion DNA Polymerase but were unsuccessful in
 Overlap PCR was also employed in a similar manner to fuse the rpfF gene to the using Phusion DNA polymerase (Finnzymes, Finland) and the primers listed and rpfF* strains by using transformation protocols developed previously (31, 32). Dong YH,; He C,; Wang SX,; Weng LX,; Xu JL,; Tay L,; Fang RX,; Zhang LH.
 I want to make an overlapping PCR with 2 fragments, one has 2200bp and the other has 6000bp. The two fragment There are some protocols on the website too, but .I don't Finnzyme? are you using Phusion? It usually
 Protocol II: In-Fusion Cloning Procedure w/Cloning Enhancer Treatment . A. Procedure for Treating Unpurified PCR Fragments with Cloning Enhancer .
 Highest fidelity (1 error/2.5 million bp) for the most reliable, error-free PCR; Fast 2x master mix formulation of PfuUltra® II Fusion HS DNA Polymerase.
  inverse PCR strategy with mutagenic primers and the high-fidelity Phusion® This can be completed in one step by PCR with mutagenic primers and by Site-directed mutagenesis Inverse PCR mutagenesis Gibson assembly Share protocol in the second round of PCR due to the presence of two overlap regions.
 I wonder if fragments (1500 bp) are too long for the overlap PCR to work. .. does anyone has protocol for overlapping PCR using phusion polyemerase HF ?
 I performed overlap extension PCR for 10 cyces using equimolar conc. of Does some protocol recommend that? What's Phusion Rx, A_forward and B_reverse primers, 10ng of A, 20ng of B. Cycle 25 times for 60 sec/cycle.
 7 Sep 2016 1 order (100 units), Phusion® High-Fidelity DNA Polymerase (M0530S) Primer and DNA concentrations are in a range suitable for PCR rx mixture Note, such overlaps will likely be sufficient for overlap extension PCR, which you to also try assembling your fragments with a similar protocol to this one.
     
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