This protocol works well from a 5 ml starting culture or a 1 ml starting culture (adjust volume accordingly). Solutions 2X YT Media 16 g tryptone 10 g yeast extract 5 g NaCl 1 ml 1N NaOH up to 1 liter with Q Ampicillin Stock (1000X) 0.15 g ampicillin 1 ml Q can be stored at 4℃ for several weeks Tetracycline Stock (1000X) 15 mg tetracycline 500 m l EtOH 500 m l Q vortex to dissolve and store at 4℃ Kanamycin Stock (1000X) 50 mg kanamycin 1 ml Q can be stored at 4℃ for several weeks 20% PEG 8000/ 2.5 M NaCl 20 g PEG 8000 14.6 g NaCl up to 100 ml with Q Procedure • Transform the appropriate plasmid construct into XL-1 Blue and plug one colony into 5 ml 2X YT + Amp + Tet. • Add 10 m l Helper phage (M13 VCS 1x10 12 pfu/ml) and incubate in the 37℃ shaker for 45 minutes. • Add kanamycin and continue to incubate in the 37℃ shaker overnight. • Pellet the cells at 4℃ for 10 minutes and transfer 1.2 ml of supernate to each of 4 eppendorf tubes. • Discard the cell pellets and add 240 m l 20% PEG/2.5 M NaCl to each eppendorf tube. • Incubate on ice for 30 minutes and spin at 4℃ for 15 minutes to pellet the phage. • Aspirate the supernate and resuspend the pellet in 400 m l Q. • Phenol/CHCl3 extract and CHCl 3 extract. • Add 0.1 volume of 3 M NaOAc, 1 m l Glycogen and 2 volumes of EtOH. Precipitate, wash and dry and resuspend in 50 m l Q. • 5-7 m l is generally enough for each sequencing reaction. |
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