• Overview
• Transformation
• Miniprep
• PCR

Miniprep

a. Transfer a single colony from the transformed E.colli to the LB medium and incubate in shaking incubator for 24 hours.

b. Centrifuge said E.colli for 15 minutes at 1500 rpm.

c. Dispose of supernatant.

d. Add 300μl of solution I and vortex.

e. Transfer the fluid into 2 EP tubes, 150μl each.

f. Add 300μl of solution II in each EP tube.

g. Add 200μl of solution III in each EP tube and mix by inverting.

h. Centrifuge both EP tubes for 10 minutes at 14000rpm

i. Transfer 550μl of supernatant to column.

j. Centrifuge at 14000rpm for 1 minute.

k. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.

l. Centrifuge at 14000rpm for 1 minute.

m. Dispose of fluid in collection tube, and add 700μl of wash buffer to column.

n. Centrifuge at 14000rpm for 1 minute.

o. Dispose of fluid in collection tube.

p. Centrifuge at 14000rpm for 1 minute.

q. Add 50μl of distilled water and centrifuge at 14000rpm for 1 minute.

r. Keep filtered solution.
solution Ⅰ 50mM glucose, 25mM Tris•Cl (pH 8.0), 10mM EDTA (pH 8.0)
solution Ⅱ 0.2N NaOH, 1% SDS
solution Ⅲ 5M potassium acetate, glacial acetic acid, H₂O