DNA precipitation (Mini-prep) 세포생물학 및 실험 2 2018-2학기 생명과학과 박태식 교수님 화요일 (1-4 교시) DNA precipitation (Mini-prep)

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

DNA purification Resuspension buffer : containing RNase solution ☞ degrade RNA Lysis buffer : containing SDS (detergent) ☞ brake the cell membrane(it can elute DNA in the cells.) Neutralization buffer : containing acetic acid (CH3COOH) ☞ neutralize negatively DNA Washing buffer A : containing remover about endonuclease ☞ remove cell toxicity Washing buffer B : containing EtOH ☞ wash other organic chemicals and others Elution buffer : containing DNase/RNase-free solution ☞ elute DNA by changing the binding. DNA-spin column : containing silica membrane ☞ can bind neutralized DNA Silica membrane

Mini prep Steps Cell suspension Cell lysis Neuralization pH Resuspension Buffer Lysis Buffer Neutralization Buffer Steps Cell suspension Cell lysis Neuralization pH Neutral ~ weak base Alkaline Subacid Color Transparent ~ pale pink Strong pink Transparent (turbid)

Protocol Pick a single colony from a freshly streaked bacterial plate and use it to inoculate 3 ml LB containing the 100 ug/ml ampicillin Incubate the culture overnight with shaking 37℃, 250 rpm.

Protocol Harvest. Harvest 2 ml of bacterial culture by centrifugation at 13,000rpm for 30 sec at RT and discard all the supernatant. ☞ 1 ml/time X 2 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!!! Incubate for 3 min at RT. 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 ℃

Protocol 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. Centrifuge at 13,000 rpm for 60 sec to dry the filter membrane.

Protocol 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 purity of dsDNA. ☞ 결과에 첨부할 것

Q. A260/A230, A260/A280 가 각각 무엇을 의미하는지 조사하시오. Discussion Q. A260/A230, A260/A280 가 각각 무엇을 의미하는지 조사하시오.