プラスミドのミニプレップは、様々なキットがあり、誰でも簡単に純度の高いプラスミドを得ることができる。とはいえ、貧乏な研究室では結構な負担ではあるので、目的により使い分けるのが妥当かと思う。例えば、動物などの培養細胞へのtransfectionに用いる場合には、ある程度以上の量を確保したいので、CsClによる超遠心法を用いている。また、sequencerにかけるには以下で延べるSDS-NaOH法により精製したプラスミドの一部をQiagenのQIAprep
Spin Miniprep Kit (50)を用いて更に精製してsequence labに出す。
以下に示すのは、比較的簡単にできるだけ多くのプラスミドを得る方法である。この方法の利点は、
1)14 mlのFalconのポリプロのチューブを3 ml TBでのovernight-cultureのculture tubeとして用い、培養後、蓋を取ってそのまま遠心チューブとして使い、最初の大腸菌ペレットの可溶化/中和を同じチューブ内で行う。これにより、培養液を1.5 mlのチューブに移す手間が省ける。
2)3 mlの培養液を処理できるので、1.5 mlチューブ2本分のプラスミドを得ることができる。
3)LBではなくTBを用いるので、大腸菌のペレットを多く処理でき、その分回収されるplasmidの量も多い。
注)欲張って3 ml以上の培養液を処理しようとすると、下記のステップ7の上澄みの処理が、1.5 ml チューブ1本では間に合わなくなる。従って、この方法では、3 mlのTBの一夜培養液を処理するのが上限である。
Equipment and tools:
Low speed
centrifuge (Sorvall or Beckman)
Microcentrifuge
14 ml round
bottom polypropylene culture tube (Falcon)
1.5
ml tube
Solutions:
Lysozyme bf.
25 mM Tris-HCl (pH8.0)
10 mM EDTA
50 mM Glucose
SDS-NaOH sol. 50ml (Maybe stored at -20 °C)
0.5 g SDS
0.4 g NaOH
3 M KAcO (pH5.2) (refer to Molecular Cloning) 250 ml
KAcO (MW. 98.15) 73.6 g
Acetic acid 28.75 ml
Phenol/chloroform
25 ml Phenol (water
saturated)
24 ml Chloroform
1 ml isoamyl alcohol
20% PEG/2.5 M NaCl (PEG 8000)
7.8 M NH4AcO
10 mg/ml DNase-free
RNase A (heat-treated. Refer "Molecular Cloning")
1. O/N culture in 3 ml
TB (use 14 ml round bottom tube).
2. Spin down (7,000
rpm, 1 min, Sorval). (Note 1)
3. Vortex bacterial
pellets in 0.2 ml Lysis bf. (10 mg/ml lysozyme). Suspend the pellet completely.
4. Incubate at room
temp for 1 min.
5. Add 0.4 ml SDS-NaOH sol, gently mix until E.
coli is lysed, and place on ice. (Solution will be clear yellow and viscous.) (Note 2)
6. Add 0.3 ml 3M KAcO (pH5.2), gently mix and
keep it on ice for 1-2 min. (Yellow and viscous solution will disappear.) (Note 3)
7. Spin down (7,500 rpm,
10 min) at 4 oC and transfer supernatant into a 1.5 ml new tube. (Note 4)
8. Add 0.6 ml phenol/chloroform, vortex, spin 10 min and transfer the upper
phase into a new tube. (Note 5)
9. Fill up the tube
with isopropanol (0.6 ml) and
mix. (Note 6)
10. Spin (10,000 g, 10 min,
maximum of microcentrifuge), and discard sup. (Note 7)
11. Wash the pellets with 1 ml of 75% EtOH, spin, and remove the sol completely by pipetting. (Note 7, 8)
12. Dissolve pellet in
0.1 ml water containing 0.1 mg/ml DNase-free
RNase A.
13. Incubate at 37 oC
for 5 min.
14. Add 0.06 ml PEG/NaCl sol and incubate on ice for 15 min. In most case, you
will see white stuffs (plasmid DNA) at this step.
15. Spin for 10 min (10,000 g). Discard sup and wash the pellets with 75% EtOH. (Note 7, 8)
16. Dissolve pellets in
0.05 ml water. (The quality of plasmids will be good enough for the most purpose. 0.001 ml for digestion to check inserts on a mini-gel.)
This protocol
yields 0.04-0.05 mg plasmid for pBlueScrip.
Option) For the digestion by restriction enzymes that require the low salt
buffer:
17. Add 0.025 ml (a half volume of 0.050 ml) 7.5 M
NH4AcO (final 2.5 M), mix, and spin at 10,000 g for 10 min and
transfer sup into a new tube.
18. Add 0.15 ml EtOH and
spin at 10,000 g for 10 min.
19. Wash the pellets with
75% EtOH and remove a trace of EtOH by pipette.
20. Dissolve the pellets
in 0.05 ml water.
Note
1) Remove caps of the culture tubes before
centrifugation. You may need to use the adaptor for 14 ml tubes. When you spin
at a higher speed, rubber adapters with centrifuge tubes may be crashed.
Note
2) When you add SDS-NaOH solution, mix the solution by spinning the tube with your hands with a
little angle one by one until the solution become
clear yellow without any dusty portion.
Note
3) Similarly, mix the solution by spinning the tube with your hands one by one until the clear yellow portion completely become white junk in nonviscous
solution.
Note
4) A small amount of junk can be transferred with the sup. It does not cause any
problem.
Note
5) 0.6 ml
Phenol/CHCl3 is a standard volume to fill the 1.5 ml tube.
Therefore, you may adjust the volume to 1.5 ml by the phenol/chloroform. Take
sup by pipetting but do not take the white junks.
Note
6) 0.6 vol of isopropanol should be enough. Usually, DNA solution at this step
is less than 0.9 ml.
Note
7) Remove the solution by pipetting to ensure
the removal of salt.
Note
8) 75-80 % ethanol solution is used for wash away the residual salt.