TransformationⅠ

Transformation & smear to LB plate 1주차 Transformation & smear to LB plate 2주차 Miniprep 3주차 Or 4주차 ①수 : Transfection ②목 : Fluorescence microscopy(RFP) & Treat TAT-cre ③금 : Fluorescence microscopy(GFP) DNA Liposomes DNA/Lipid complex

DNA purification Introduction Resuspension buffer : containing RNase solution Lysis buffer : containing SDS(detergent) Neutralization buffer : containing acetic acid Washing buffer A : containing remover about endonuclease Washing buffer B : containing EtOH Elution buffer : containing DNase/RNase-free solution DNA-spin column : containing silica membrane

Principle of DNA purification Introduction Principle of DNA purification RNase solution ☞ degrade RNA SDS(detergent) ☞ brake the cell membrane(it can elute DNA in the cells.) acetic acid(CH3COOH) ☞ neutralize negatively DNA remover about endonuclease ☞ remove cell toxicity EtOH ☞ wash other organic chemicals and others DNase/RNase-free solution ☞ elute DNA by changing the binding. silica membrane ☞ can bind neutralized DNA Silica membrane

Mini prep Introduction 6. Wash 1. Harvest 2. Resuspend 7. Elute 3. Lysis 4. Precipitate 5. Bind

Introduction Mini prep

Mini prep Materials & Methods Pick a single colony from a freshly streaked bacterial plate and use it to inoculate 5 ml LB plus an 5 ul of the 100mg/ml ampicillin (the final concentration, 100 ug/ml ampicillin). Incubate the culture overnight with shaking 37℃, 250 rpm.

Mini prep Materials & Methods Harvest. Harvest 5 ml of bacterial culture by centrifugation at 13,000rpm for 30 sec at RT and discard all the supernatant. ☞ 1 ml/time X 3 times repeat Resuspend. Resuspend the pellet in 250 ul of Resuspension Buffer(RF), vortexing until no clumps of the cell pellet remain. Lyse. Add 250 ul of Lysis buffer(LB) to resuspended cells. Close tube and gently mix by inverting the tube 5-6 times. Do not vortex!!! Precipitate. Add 350 ul of Neutralization buffer(NB) and gently mix by inverting the tube 5-6 times. Do not vortex!!! And incubate in ice for 5 min. Centrifuge at 13,000 rpm for 10 min at 4 ℃ ★Labeling!! ★

Mini prep Materials & Methods Bind. After centrifuge, transfer supernatant 700 ul promptly into the column. And then incubate at RT for 5 min. Do not transfer with white pellet!! Centrifuge at 13,000 rpm for 60 sec. Discard filtrate in collection tube. And then place the spin column back in the same collection tube. Wash 1. Add 500 ul of Washing buffer A(WA) and centrifuge at 13,000 rpm for 60 sec. Discard filtrate in collection tube. And then place the spin column back in the same collection tube. Wash 2. Add 700 ul of Washing buffer B(WB) and centrifuge at 13,000 rpm for 60 sec. Discard filtrate in collection tube. And then place the spin column back in the same collection tube.

Mini prep Materials & Methods Centrifuge at 13,000 rpm for 60 sec to dry the filter membrane. Elute. Put the column into a new E-tube. Add 50 ul of Elution buffer(EB) to the upper membrane of the column, and let it stand for 5 min. Centrifuge the tube assembly at 13,000 rpm for 60 sec. Check the concentration and purify of dsDNA.

Result Result - DNA purity, concentration ★ report 제출(5.25일 실험 수업 시작 전에 제출)

Discussion Q. DNA isolation kit를 사용하지 않고 분리하는 방법을 하나 이상 서술하시오

3주차 실험 할 조 : 2조 3조 4조 4주차 실험 할 조 : 6조 1조 8조