Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/03

Today's project is...

Main project page Previous entry      Next entry

Overview Performing Ligase Cycling Reaction (LCR) today. See instructions here. Methods Prep Bridge Oligos Make 300 nM working solutions of the dsDNA oligo bridges: 3 µL forward strand (100 µM) 3 µL reverse strand (100 µM) 94 µL H2O Prep DNA fragments Dilute purified DNA from Phusion PCR to 30 nM: Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL DNA fragment Length (bp) measured ng/µL purified DNA volume (µL) final volume (µL) HA1 100 149 0.65 50 HA2 100 127 0.77 50 EM7-ZeoR 442 235 1.83 50 HPK-CFP 1972 24 80.11 50 Since HPK-CFP fragment is at too low a concentration, will use it straight instead. PNK Reaction Use polynucleotide kinase (PNK) to add 5' phosphate groups to DNA fragments. Use 2.0 μL of each diluted dsDNA per 10 μL PNK reaction Use 2.5 µL of HPK-CFP dsDNA NOTE: All DNA to be treated with PNK goes in the same tube! Reagent Volume Clean PCR or dsDNA 8.5 μL 10x T4 Ligation buf (NEB) 1.0 T4 PNK (NEB) 0.5 dH2O 0 μL   10.0 μL Incubate at 37°C/ 30 min. Heat-inactivate PNK at 65°C/ 20 min. Ligase Cycling Reaction In a PCR tube, set up the following reaction. Use 3 µL of each dsDNA oligo bridge (9 µL total). Reagent Volume PNK DNA 10 μL dsDNA Oligo Bridge 9.0 10X Ampligase Buffer 3.0 DMSO 2.4 Betaine (5 M) 2.7 Ampligase 1.0 dH2O 1.9 μL   30.0 μL Thermal cycler program: 2 min at 94 °C 10 s at 94 °C 30 s at 55 °C 60 s at 66 °C Repeat steps 2-5 x50 times hold at 4 °C