분자생물학실험 | Midi-preparation of plasmid DNA

 

 

 

Midi-preparation

mini-preparation 과 실험의 원리가 똑같으나, 실험자가 원하는 gene의 수를 늘리기 위해, 즉 bacteria를 증폭시키기 위해 scale을 늘린 실험.

mini-preparation은 5∼10㎍을 준비하는데 비해, midi-preparation은 ∼100㎍정도의 양까지 증폭시킨다.

 

 

실험 방법

Step 1 : 선처리

1) inoculate two single well isolated colonies into two 1.5 ㎖ LB+amp (working concentration 50 ㎎/㎖) culture tubes (a colony/clone per tube)

 

2) incubate for approximately 8 hours at 37℃ with vigorous shaking (∼300 rpm)

 

3) add the seed culture (1.5 ㎖ LB+amp) to 50 ㎖ of LB medium+amp (working conc. 50 ㎎/㎖) (dilution range 1/33.3 ∼ 1/1000, usually, 1/33.3, 1/250, 1/500)

 

4) incubate for 12∼16 hours at 37 ℃ with vigorous shaking (∼300 rpm)

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Step 2

1) harvest the bacterial cells by centifugation at 5000 rpm (5000 g) for 15 min at 4 ℃

 

2) resuspend the bacteria in 4 ㎖ Buffer P1

 

3) add 4 ㎖ Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4∼6 times, and incubate at room temperature for 5 minutes

 

4) add 4 ㎖ of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4∼6 times, and incubate on ice for 15 minutes

 

5) spin at 13000 rpm (∼20000 g) for 30 min at 4 ℃

 

6) take supernatant containing plasmid DNA promptly

 

7) equilibrate a QIAGEN-tip 100 by applying 4 ㎖ Buffer QBT, and allow the column to empty by gravity flow

 

8) apply the supernatant from step 6 to the QIAGEN-tip and allow it to enter the resin by gravity flow

 

9) wash the QIAGEN-tip with 10 ㎖ Buffer QC twice

 

10) elute DNA with 5 ㎖ Buffer QF

 

11) aliquot 900 ㎕ of eluted DNA to each microtube

 

12) precipitate DNA by adding 630 ㎕ (0.7 volume) of room-temperature Isopropanol (2-propanol) to the eluted DNA

 

13) mix, spin immediately at 13000 rpm (15000 g) for 30 min at 4 ℃ and carefully decant the supernatant

 

14) wash DNA pellet with 2 ㎖ of room-temperature 70 % ethanol, and spin at 13000 rpm for 10 min

 

15) carefully decant the supernatant without disturbing the pellet

 

16) air-dry the pellet for 5∼10 min and redissolve the DNA in a 10 ㎕ of 10 mM Tris-Cl, pH 8.5

 

17) measure DNA concentration with spectrophotometer

 

18) agarose gel electrophoresis

 

 

 

[분자생물학실험]Midi-preparation of plasmid DNA 레포트