Bacterial plasmids are commonly used gene delivery vehicles in recombinant DNA technology. Alkaline lysis is a widely employed method for small-scale plasmid DNA preparation.
Experimental Steps
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Filter the bacterial culture, transfer the filtrate to a 1.5 mL centrifuge tube, centrifuge at 6000 r/min for 2 minutes, and discard the supernatant.
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Add 100 μL of pre-chilled Solution I to the remaining bacterial pellet. Vortex vigorously to fully resuspend the pellet, and then leave at room temperature for 5 minutes.
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Add 200 μL of Solution II, close the lid, and gently invert the centrifuge tube several times (do not vortex). Leave at room temperature for 5 minutes.
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Add 150 mL of Solution III, gently mix to homogenize, and leave at room temperature for 5 minutes.
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Centrifuge at 12,000 r/min (13,400 × g) for 10 minutes. Transfer the supernatant to a new centrifuge tube.
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Add an equal volume of phenol/chloroform/isoamyl alcohol (approximately 350 μL). Shake to mix the organic and aqueous phases, then centrifuge at 12,000 r/min for 10 minutes. Transfer the supernatant to a new centrifuge tube.
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Add 2 volumes of absolute ethanol, mix well, and leave at -20°C for 30 minutes.
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Centrifuge at 12,000 r/min for 10 minutes. Carefully discard the supernatant. Add 1 mL of 70% ethanol to wash the pellet. Centrifuge at 12,000 r/min for 2 minutes and discard the supernatant.
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Place the centrifuge tube with its lid open in a clean bench to air-dry the ethanol.
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Add 2 μL of RNase A solution and 20 μL of TE buffer to dissolve the pellet. Incubate at 37°C for 20 minutes, then store at -20°C.
Featured Products
J&K Scientific provides reagents, pipettes and consumables for plasmid DNA extraction via alkaline lysis.
Solution I (GTE Solution)：
- 226162 Tris(hydroxymethyl)aminomethane, 99.5%, extra pure, CAS:77-86-1
- 932641 Ethylenediaminetetraacetic acid, 99%, reagent grade, CAS:60-00-4
- 936778 D-(+)-Glucose, 99%, for bio, CAS:50-99-7
Solution II ：
- 904646 Sodium hydroxide, 96%, for analysis, flaky, CAS:1310-73-2
- 105893 Sodium dodecyl sulfate, 99%, for biochemistry, CAS:151-21-3
Solution III ：
- 991956 Acetic acid, 99.5%, for analysis, CAS:64-19-7
- 978296 Potassium acetate, 99%, for analysis, CAS:127-08-2
LB Medium ：
- 716769 Yeast extract powder, CAS:8013-01-2
- 908606 Sodium chloride, 99.5%, molecular biology grade, CAS:7647-14-5
RNase A Solution ：
- 139924 Ribonuclease A, from bovine pancreas, ≥50 Kunitz units/mg, CAS:9001-99-4
TE Buffer ：
- 226162 Tris(hydroxymethyl)aminomethane, 99.5%, extra pure, CAS:77-86-1
- 932641 Ethylenediaminetetraacetic acid, 99%, reagent grade, CAS:60-00-4
Other Reagents ：
- 226162 Ethanol, 99.7%, reagent grade, anhydrous, CAS:64-17-5
- 923740 Isopropanol, 99.7%, for synthesis, CAS:67-63-0
Small Instruments ：
- 1122421 Single-channel mechanical pipettor, Volume range: 0.5 - 10 μL, Increment: 0.1 μL
- 954106 Single-channel mechanical pipettor, Volume range: 20 - 200 μL, Increment: 1.0 μL
- 1122427 Single-channel mechanical pipettor, Volume range: 100 - 1000 μL, Increment: 5.0 μL
Basic Consumables ：
- 915664 Centrifuge tubes, 1.5 mL, clear, 500 tubes/pack
- 920067 Pipette tips, 10 μL, clear, 1000 tips/pack
- 970956 Pipette tips, 200 μL, yellow, 1000 tips/pack
- 672890 Pipette tips, 1000 μL, blue, 1000 tips/pack
艳 李
