Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/10/28

Phusion PCR of parts for LCR

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Overview We're using the Phusion polymerase to create excess template in anticipation of performing ligase-cycling reaction (LCR) in the near future. This will allow us to incorporate a zeocin resistance (ZeoR) gene upstream of the HPK-CFP fluorescent reporter. Methods Reaction volume: 100 µL (split into 4 tubes to be pooled later) Reagent Stock Volume used (µL) HF Buffer 5x 20 dNTP mix 10 mM 2 Primer (for) 100 µM 1 Primer (rev) 100 µM 1 Template 50 ng/µL 1 Phusion 20/µL 1 H2O - 74 Total 100 Reactions: Reaction Forward primer Reverse primer template HA1 (1) P40 P120 DBN007 EM7-ZeoR (2) P121 P122 MV8 HPK-CFP (3) P123 P124 DBN007 HA2 (4) P125 P41 DBN007 All primers have annealing temperature at 60°C Thermal cycler program: STEP TEMP TIME Initial Denaturation 95°C 30 seconds 30 Cycles 95°C 10 seconds 60°C 20 seconds 72°C 60 seconds Final Extension 72°C 10 minutes Hold 4°C infinite