Procedure for Plasmid Purification:
1. Grow Bacteria:
a. Place 5 ml of LB with 50 µg/mL Kanamycin (or 100 µg/mL
Ampicillin) in glass test tube for each colony in which you want
to purify the plasmid.
b. Take out a new LB/KAN plate and make a grid that is the same
number as the tubes for the colonies you are starting.
c. Take a 6-inch wooden stick and pick the whole colony from
your plate and the touch it to the corresponding number on the
“grid” plate (you will not be able to see anything on the plate
but the mark the stick made-it will grow just fine).
d. Then take the remaining material on the stick and mix it in the
liquid LB/KAN test tube. Take the stick out and place in the
used stick container (DO NOT THROW AWAY).
e. Place tubes in the 37°C shaking incubator (220 rpm) and let
grow overnight.
f. Proceed to plasmid purification section 2.
2. Purification of Plasmid (Boiling method):
a. For each culture label 3 1.5-mL tubes.
b. Transfer culture into microcentrifuge tubes using a pipet.
c. Centrifuge at maximum speed for 5 minutes.
d. Pour off the supernatant into waste container by centrifuge.
Remove as much of the liquid as possible by dabbing on a
paper towel (this will be demonstrated by lab instructor).
e. Add 700 µL of Sucrose Lysis Buffer to one tube and
resuspend by pipetting up and down (to prevent frothing do not
pipet up and down all the liquid in the tube).
f. Transfer resuspended cells to next 1.5-mL tube and resuspend
pellet. Then transfer resuspended cells to last 1.5-mL tube and
resuspend. Make sure all the pellet is completely resuspended
before proceeding to next step.
g. Add 50 µL of 10 mg/mL Lysozyme (LYS) to tubes and invert
tubes to mix.
h. Incubate on your bench at room temperature for 5 minutes.
i. Heat tubes at 99°C for 2 minute in a heat block (make sure
that well in heat block are filled with water to create proper
transfer of heat).
j. Centrifuge at maximum speed for 15 minutes.
k. Remove pellets with a toothpick and discard pellets along with
toothpick (SAVE THE SUPERNATANT!!!).
l. Add 80 µL of 3 M NaOAc and 440 µL of isopropanol to the
supernatant in each tube to precipitate the DNA.
m. Invert the tubes to mix and then incubate at room temperature
on the bench for 5 minutes.
n. Centrifuge at maximum speed for 5 minutes.
o. Discard supernatant (make sure that the pellet is not lost when
pouring off the supernatant) and dab on paper towel.
p. Add 1 mL 70% ethanol to each tube to wash the salt off the
plasmid pellets.
q. Centrifuge at maximum speed for 5 minutes.
r. Discard the supernatant and dab on paper towel.
s. Centrifuge at maximum speed for 20 seconds and then remove
liquid from the tubes with p200 pipet.
t. Centrifuge at maximum speed for 20 seconds and then remove
liquid from the tubes with a p10 pipet.
u. Allow pellets to completely dry for 10-15 minutes with the
caps open at room temperature on bench.
v. Add 50 µL of 1X TE (pH 8.0) to each tube and let sit on bench
for 5 minutes.
w. Resuspend pellet by pipetting up and down (set pipet at 25 µL
so you do not take up al liquid and create bubbles).
x. DNA can be stored at -20°C