MINIPREP PROTOCOL: Isolating Plasmid DNA
1) Make NaOH/SDS solution*** (If not available).
- You will need 200ul per sample, follow the table below:
Stock
To make 5mL
Final Concentration
10M NaOH
0.1mL (100uL)
0.2M
20% SDS
0.25mL (250uL)
1%
DsH2O
4.65mL
***Safety: The NaOH/SDS solution is caustic so handle carefully.
2) Gently vortex bacterial broth solution and micropipette 1000uL into labeled 1.5mL tubes.
-The vortexing is to make sure that the bacteria are not in clumps.
3) Centrifuge at 13,000 rpm for 1 minute; remove all the supernatant using a micropipette and
flash spin to collect.
4) Repeat steps 2 -3 using the same 1.5 mL tubes and culture tubes as necessary.
-You want to collect as many bacteria as possible from one bacterial culture tube in the
labeled 1.5mL tube. It is very important that you DO NOT mix the broth from two
different culture tubes!
5) Remove the remaining growth media using a micropipette.
6) Add 100uL TE pH 8.0 to the labeled 1.5mL tube and vortex briefly.
7) Add 200uL of the NaOH/SDS solution; mix by inverting.
8) Put on ice for ~5minutes.
9) Add 150 uL of KOAc (Potassium Acetate pH~4.8) and mix by inverting several times.
10) Put on ice for ~5 minutes.
11) Centrifuge at 13,000 rpm for 2 minutes.
12) Transfer the supernatant to new, labeled 1.5 mL tubes.
- When transferring be careful to not get any precipitate into new tubes.
13) Add 900 uL of 100% ETOH (needs to be cold) and mix by inverting.
14) Incubate for 2 minutes, centrifuge at 13,000 rpm for 1 minute and pour off the supernatant.
- The cold ethanol precipitates the plasmid DNA
15) Add 500 uL of 70% ETOH, mix by inverting and centrifuge at 13,000rpm for 30seconds.
*Written by Vinh Vu, modified from “Short Protocols in Molecular Biology” as employed by the Silliker lab
Last Updated Feb/4/12
16) Pour off the supernatant and flash spin.
17) Use a micropipette to take out the remaining alcohol.
-It is important to take out as much ethanol as possible because it can interfere with
downstream sequencing reactions.
18) Air-dry the pellets to remove residual ETOH in the hood.
19) Suspend in 50uL TE pH 8.0.
*Written by Vinh Vu, modified from “Short Protocols in Molecular Biology” as employed by the Silliker lab
Last Updated Feb/4/12