Quick BAC Mini-prep Protocol
1.Start overnight culture in a 2 ml epi-tube. (Puncture the cap for aeration.) Incubate in Eppendorf ThermalMixer at 37 °C, 900 rpm for 14-16 hours.
2.Spin down at 13200 rpm for 1 minute and discard the supernatant.
3.Add 200μl of buffer P1 and mix up by Eppendorf MixMate at 1300 rpm for about 10 minutes, and check if there are cells still attaching on the tube.
4.Add 200μl of buffer P2 and mix by Eppendorf MixMate at 1300 rpm for 3-5 seconds, repeat 3 times. (or gently invert 3 times in hands)
5.Add 200μl of buffer P3 and mix by Eppendorf MixMate at 1300 rpm for 3-5 seconds, repeat 3 times. (or gently invert 3 times in hands)
6.Gently invert the tubes three times.
7.Spin down the white lysate at highest speed for 10-20 minutes.
8.Set up new 1.5 ml epi-tubes in a rack with lid and add 500μl of iso-propanol in the tube.
9.Transfer the clear supernatant into the new tubes which have iso-propanol.
10.Cover the rack with the lid and mix by very strong up-and-down shaking in hands for 5-6 times.
11.Spin at highest speed for 20 minutes.
12.Discard the supernatant and invert the tubes on a piece of tissue.
13.Put the tubes on a rack and gently add 1ml of 70% ethanol.
14.Discard the ethanol and invert the tubes on a piece of tissue.
15.Clean the inner wall of the tube with a self-made tissue roller or a cotton stick.
16.Dry the DNA pellet in the ThermalMixer at 45 °C for 10-20 minutes.
17.Resolve the DNA pellet in 13ul of digestion mixture, and incubate for about 3 hours.
18.Run the samples on the gel less than 60 Voltage.
19.Make a photo under UV light.
P1
25 mM Tris-HCl (pH8.0)
10 mM EDTA
50 mM Glucose
115℃,30 min, Sterilize
Keeping at 4℃
2 mL RNase A (20 mg/mL) per 50 mL P1 when use
P2
200 mM NaOH
1% (W/V) SDS
Keeping at room temperature and no longer a month
P3
3 M KOAc
5 M CH3COOH
115℃,30 min, Sterilize
Keeping at 4℃
This protocol is also used for high copy plasmid mini-prep. 17. Resolve the DNA pellet in 20-40ul of EB buffer (depend on the copy number) and use 4ul for restriction analysis.
关键词：质粒提取 Protocol