11 Plasmid miniprep E Coli transformation Gel electrophoresis

생유기화학실험 11주차 Plasmid mini-prep E. Coli transformation & Gel electrophoresis www. company. com

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Plasmid preparation – alkaline lysis www. company. com

Alkaline lysis solutions Solution I 50 m. M glucose 25 m. M Tris-Cl (p. H 8. 0) 10 m. M EDTA (p. H 8. 0) Rnase A Solution II 0. 2 N Na. OH 1% (w/v) SDS Adjust osmotic pressure Adjust p. H Prevent DNA degradation Catalyze RNA degradation Denaturation of DNA Denaturation of protein Disrupt cell membrane Solution III 3 M KOAc 11. 5% (v/v) Acetic acid Renaturation of DNA www. company. com

Materials & Methods - Alkaline lysis solutions Overnight cultured E. coli Table-top centrifuge TE Buffer (p. H 8. 0, 20 ug/m. L RNase A) Ethanol (100 %, 70 %) Spectrophotometer www. company. com

Materials & Methods b l 2 1 1. Resuspend the pellet in 100 u. L of ice-cold Sol I. 2. Add 200 u. L of fresh Sol II and invert five times (DO NOT SHAKE VIGOROUSLY!!). 3. Store the tube on ice. 4. Add 150 u. L of ice-cold Sol III and invert several times. Store the tube on ice 3 -5 min 5. Centrifuge for 5 min at 4 o. C. Transfer the supernatant in a new tube. www. company. com

Materials & Methods 6. Add 2 volume of Et. OH at RT. Vortex and store at RT for 2 min. 7. Centrifuge for 5 min at 4 o. C to collect the DNA. 8. Remove the supernatant and dry the tube. 9. Add 1 m. L of 70 % Et. OH and invert several times. 10. Centrifuge for 2 min at 4 o. C. 11. Remove the supernatant and dry the tube at RT. 12. Dissolve the DNA in 50 u. L of TE buffer. www. company. com

Agarose gel electrophoresis www. company. com

Materials & Methods - 1% agarose gel TAE Buffer (p. H 8. 0) 6 X loading dye DNA sample Size marker Ethidium Bromide (CAUTION!!) www. company. com

Next week Protein expression (T 7 RNA Polymerase) 예 비 레 포 트 : Lac operon? Induction? IPTG? Lysis buffer의 각 Reagent들의 역할? www. company. com