从Qiagen公司试剂盒整理的BAC dna方法

从Qiagen公司试剂盒整理的BAC dna方法，希望对大家有用。

BAC DNA Isolation

1. Randomly pick white colonies with sterilized toothpicks and inoculate each into
2 mL of LB containing 12.5 μg/mL chloramphenicol in a sterile 15-mL culture
tube. Grow the cells at 37°C overnight with vigorous shaking.
2. Transfer each cell culture (about 1.5 mL) into a microcentrifuge tube and collect
cells at 16,000g (at room temperature or 4°C) for 10 min; remove supernatant.
3. Add 200 μL of P1. Mix the tubes with a vortex to resuspend pellets at room
temperature.
4. Add 200 μL of P2. Mix the contents gently but thoroughly by inverting the tubes
3 to 4 times. Stand the tubes at room temperature for not more than 3 min.
5. Add 200 μL of P3. Mix the contents gently but thoroughly by inverting the tubes
3 to 4 times. Stand the tubes on ice for 15 min.
6. Centrifuge the samples at 16,000g (at room temperature or 4°C) for 30–40 min.
7. Carefully transfer about 550 μL of each supernatant to a new microcentrifuge
tube containing 400 μL of isopropanol. Mix the contents gently.
8. Centrifuge the samples at 16,000g (at room temperature or 4°C) for 30 min.
9. Remove the supernatant. Add 400 μL of 70% ethanol and centrifuge the samples
at 16,000g for 10 min to wash the DNA pellets.
10. Remove the supernatant carefully with a pipet. Air-dry the DNA pellets, and
resuspend in 60 μL of TE buffer, pH 8.0.

Buffer                                               Composition                                      Storage
BufferP1                                         50 mM Tris·Cl, pH8.0;                            2–8°C,
(resuspensionbuffer)                        10 mMEDTA;                                     after addition
                                                      100 μg/ml RNaseA                               of RNase A

Buffer P2 (lysisbuffer)                      200 mM NaOH, 1%SDS                      Room temp.

BufferP3                                        3.0 M potassiumacetate,                      Room temp.
(neutralizationbuffer)                       pH5.5                                                   or 2–8°C

Preparation of LB medium
Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 800 ml distilled water. Adjust
the pH to 7.0 with 1 M NaOH. Adjust the volume to 1 liter with distilled water. Sterilize
by autoclaving.