plasmid prep kits: good & bad?
nishir at ohsu.edu
Wed Dec 28 15:11:30 EST 1994
Why buy a kit when the following is so easy and gives great yields?
Fast Plasmid Mini-Prep
from Zhou et al, BioTechniques vol 8, no 2
Solution A (good for about 2 weeks)
To make 100 mls:
0.8 g NaOH (0.2 M)
10 mls 10% SDS (1%)
Solution B (keep at 4¡C)
To make 1 liter:
285 mls dH2O
115 mls HOAc
600 mls 5M KOAc
1. Start Mini-prep the day before by inoculating 4 mls of LB/amp with
one colony picked from your agar plate. Shake overnight at 37¡C.
2. Pour 1.5 ml of the overnight culture into a 1.5 ml Ependorf tube
(fill to nearly top; leave some room to close cap). Spin for 1 min in
microfuge to pellet cells.
3. Decant supernatant by pouring into bleach, leaving 50-100µl of
medium behind on the cell pellet. Vortex vigorously at high speed to
resuspend cells in residual medium.
4. Add 300 µl of Solution A, cap tube immediately and shake vigorously
up and down. DO NOT VORTEX. Put on ice after shaking until you have
added soln II and shaken all your other tubes. Try not to leave
everything in soln II for any longer than 5 min because chromosomal DNA
will start to degrade and co-precipitate with the plasmid in later
steps. Solution will clear.
5. Add 150 µl of cold Solution B to each tube, cap and shake vigorously
up and down. White stuff will precipitate.
6. Spin in microfuge 3 min to pellet cell debris and chromosomal DNA;
transfer supernatant to new 1.5 ml Ependorf tube, being careful to
avoid transferring any white gunky stuff.
7. Add 1 ml of 100% ethanol that has been stored at -20¡C (look on top
shelf of DATA). Vortex.
8. Spin 5 min in microfuge to pellet plasmid DNA and cellular RNA.
9. Pour off supernatant and add 1ml of cold (4¡C) 70% ethanol. Vortex.
Spin 5 min in microfuge.
10. Remove supernatant. Wick out last drop of ethanol with kimwipe.
Let pellet dry on lab bench (turn tube upside down or on its side).
This will take 20-30 min. You will know when the pellet is dry because
the white pellet will become clear. DonÕt over-dry or the DNA will be
hard to resuspend.
11. Resuspend pellet in 50µl of TE, For restriction digests cut 10µl
of DNA for 1-2 hrs in a final volume of 35 µl with 10U of restriction
enzyme. DonÕt forget to add 1µl of RNase A (1 mg/ml) along with the
Dept. Cell Biology & Anatomy
Oregon Health Sciences University
Portland Oregon 97201
**that's Orygun, NOT Ora-Gone**
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