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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 ml EtOH
500 ml 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 ml Helper phage (M13 VCS 1x1012 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 ml 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 ml Q.
• Phenol/CHCl 3 extract and CHCl3 extract.
• Add 0.1 volume of 3 M NaOAc, 1 ml Glycogen and 2 volumes of EtOH.
Precipitate, wash and dry and resuspend in 50 ml Q.
• 5-7 ml is generally enough for each sequencing reaction.
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PREPARATION OF PLASMID D