Plasmid DNA extraction can: (1) rapid purification of plasmids; (2) for molecular biology experiments such as sequencing, in vitro transcription and translation, restriction enzyme digestion, bacterial transformation, etc. The principle of the experimental method Plasmids are mostly double-stranded, circular DNA molecules, which are auxiliary genetic units that replicate and inherit independently of the bacterial chromosome. Plasmids are the most important DNA vectors for molecular biology experiments and genetic engineering to improve species.
The basic steps of plasmid extraction are divided into three steps:
â‘ Cultivation of bacteria and amplification of plasmids
â‘¡Bacterial lysis
â‘¢ Purification of plasmid DNA
E. coli
Reagents, kits Tris-Hcl EDTA glucose NaOH KAac NaAc isopropanol lysozyme phenol: chloroform absolute ethanol LB medium
Instruments, consumables, ultra-clean workbench, incubator, shaker, constant temperature water bath, tabletop centrifuge, liquid extractor, low temperature refrigerator, frozen vacuum dryer, electrophoresis instrument, horizontal electrophoresis tank, ultraviolet observer
Experimental Step 1. Materials and reagent preparation
1. Material: E. coli.
2. Apparatus: ultra-clean workbench, incubator, shaker, constant temperature water bath, tabletop centrifuge, one set of liquid extractor, low temperature refrigerator, freeze vacuum dryer, electrophoresis instrument, horizontal electrophoresis tank, and ultraviolet observer.
3. Reagents:
(1) Solution I: 25 mM Tris-Hcl (pH7.4), 10 mM EDTA (pH8.0), 50 mM glucose, autoclaved, and stored at 4 ° C.
(2) Solution II: 0.2 M NaOH, 1% SDS is now used.
(3) Solution III: 5 N KAc pH4.8, autoclaved, stored at 4 ℃.
(4) 3 M NaAc pH5.2, autoclaved and stored at 4 ℃.
(5) Isopropyl alcohol, lysozyme (8 mg / ml), phenol / chloroform, absolute ethanol, 70% ethanol, LB medium, electrophoresis reagents.
2. Operation steps
1. Bacterial propagation: LB medium, 2 ml / 20 ml, 37 ° C, 200 rpm, shake overnight.
2. Centrifuge for 10 min, 5 000 rpm, 4 ° C; discard the liquid.
3. Wash the pellet (cell cells) with pre-cooled TES buffer, centrifuge for 10 min, 5 000 rpm, 4 ° C, add pre-chilled 1 ml Solution I, and ice bath for 10 min.
4. Resuspend, add 150 ul of lysozyme mother liquor, and leave at room temperature for 5 min.
5. Add 1.2 ml Solution II and ice bath for 5 min.
6. Add 0.9 ml of pre-chilled potassium acetate, mix well, centrifuge for 10 min, 12 000 rpm, 4 ° C.
7. Add 1.5 ml of isopropanol and place in the refrigerator at -20 ° C for 15 min.
8. Centrifuge for 10 min, 12 000 rpm, 4 ° C.
9. Take the pellet and suspend it in 400 ul TE buffer.
10. Add 40 ul, 3 M NaAc.
11. Phenol / chloroform, extraction, ethanol precipitation.
12. Centrifuge for 10 min, 12 000 rpm, 4 ° C.
13. Take the precipitate, freeze-dry it, and resuspend in 50 ul TE buffer.
14. Electrophoresis to detect the quality of extracted DNA.
Vintage Tissue Box,Vintage Roses Tissue Box,Vintage Tissue Box Holder,Vintage Tissue Box Cover
ZHEJIANG NEW WOOD MATERIAL TECHNOLOGY CO.,LTD , https://www.newmaterialfurniture.com