Bacteria culture apparatus and reagents including incubator shaker, sterile 2L flask, test tube, media, antibiotics and miniculture
Qiagen Maxiprep Kit (Cat# 12163). Place buffer P3 on ice to cool. If the box is first time opened, make sure the RNase A is added into buffer P1 and store at 4oC
1 L centrifuge bottles (Beckman)
250 ml centrifuge bottles (Beckman)
50 ml centrifuge tubes (Fisher) autoclaved
Centrifuge and ultracentrifuge
Ice
Procedure
Start 5-10 ml culture (with appropriate antibiotics) from a single colony in the morning.
At about 5'o clock the afternoon, inoculate 0.5 ml each in two 500 ml media with appropriate antibiotics in 2L flasks and grow the culture at 37oC overnight with vigorous shaking at 300 rpm.
Next morning, put buffer P3 on ice. harvest cells by centrifugation at 3000 rpm for 15 min at 4oC in 1000ml centrifuge bottles.
Decant supernatent and resuspend pellets from 1000 ml culture in 30 ml buffer P1 with RNase A. Transfer to 250ml centrifuge bottle.
Add 30 ml buffer P2 while swirling the bottle gently to avoid forming clamps and incubate at room temperature for 5 min.
Add 30 ml chilled buffer P3 while swirling the bottle gently then incubate on ice for 20-30 min.
Centrifuge at 25,000 X g for 30min at 4oC. (At 20,000 X g it would not be able to seperate the lysate and the pellet clearly. 28,000 ~ 30,000 xg works well. ).
Transfer the supernatant into 2 X 50 ml centrifuge bottles, centrifuge at 28,000xg for 30min at 4C.
Usually the second centrifugation with 250ml centrifuge bottle at the same speed does not help.
Equilibrate the Qiagen-tip with 10 ml buffer QBT.
Apply the supernatant from step 7 to the QIAGEN-tip and allow it to enter the resin and go through by gravity flow.
Wash the tip with 2X30ml Buffer QC.
Elute DNA with 15 ml Buffer QF to a fresh 50ml centrifuge tube.
Precipitate DNA by adding 10.5ml (~11ml) room-temperature isopropanol to the eluted DNA solution. Mix and centrifuge immediately at 25,000 X g for 30min at 4C. Carefully decant the supernatant.
Wash DNA pellet with 5ml of room-temperature 70% ethanol and centrifuge at 25,000 X g for 15min. Carefully decant the supernatant without disturbing the pellet.
Air-dry the pellet for 30min. Redissolve the DNA in a suitable volume (0.5-1.0ml) of 10mM Tris, pH8 buffer. Store at 4oC.
OR dissolve in 5ml buffer followed by Phenol/CIA-Ethanol precipitation.