Haynes Lab:Notebook/Short Projects/2015/06/02

Haynes Lab - Short Projects

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Ben Nyer - 02/06/15 Cloning gRNAs into pSPgRNA for dCas9-HT binding to luc gene All 7 cultures showed growth in LB-Amp liquid medium. Performed plasmid minipreps and eluted in 50 µL DNase-free H2O. Nucleic acid quantification: Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280 g025 1 756.7 18.69 1.93 g025 2 670.6 13.37 1.93 g031 1 558.2 10.53 1.93 g034 1 617.4 8.81 1.92 g034 2 570.4 16.40 1.93 g048 1 660.7 9.80 1.92 g048 2 565.5 6.63 1.91 Restriction digest: Part Conc. (ng/µL) Vol. for 600ng (µL) Vol. water (µL) g025 1 756.7 0.8 25.2 g025 2 670.6 0.9 25.1 g031 1 558.2 1.1 24.9 g034 1 617.4 1.0 25.0 g034 2 570.4 1.1 24.9 g048 1 660.7 0.9 25.1 g048 2 565.5 1.1 24.9 Reagent Volume (µL) DNA + water 26 10x FD Buffer (Green) 3 ApaLI 1 Total 30 Incubate at 37°C for 10 minutes, then run total volume on the gel. Expected size fragments are 1.2kb and 2.0kb. Digest Results shadow effect probably because the table was bumped during imaging. Sequencing Submit 200ng DNA + 1 µL of 10 µM primer (René's P139 primer works) in 10 µL total volume (make up difference with DNase-free H2O). g025-1 g025-2 g031-1 g034-1 g034-1 g034-2 g048-1 g048-2