This kit is good for purifying
plasmid preps for routine analytical analysis, restriction
digestion, PCR reactions and including transfection. Reagents
are enough for about 75 midi preps. Midi preps for following
protocol will take almost same time as mini preps. Also,
there is no significant cost difference in growth media
for midi verses mini preps. Therefore, it is recommended
to use this kit for making midi preps only-the yield will
be sufficient for the screening of positive colonies and
for subsequent molecular biology applications. This protocol
is designed to make midi plasmid preparations which could
give as high as ~75 µg of high copy plasmid DNA.
You can scale down the procedure for mini preps.
CONTENTS:
Resuspension Solution
30 ml
Lysis Solution 30 ml
Neutralization Solution 30 ml
Precipitation Solution 100 ml (in brown bottles)
Ethanol wash 20 ml (10X)
Complimentary Solutions:
1X TE (Tris EDTA Buffer) 30 ml
10 X TE (Tris EDTA Buffer) 30 ml
Gel Running Buffer (50 X TAE) 50 ml
(Running electrophoresis buffer: 0.5 X)
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Midi Preps:
1. Grow ~10 ml bacterial culture for 12-15 hrs with
vigorous shaking at 37?C. 50 ml capacity disposable
polypropylene tubes usually work well for growth of
12 ml of bacterial cultures. Pellet the cells by centrifugation
at 3000 rpm (2200g) for 15 minutes. Discard the supernatant.
Spin the tube again for 1 minute to remove residual
media.
2. Re-suspend the pellet in 300 µl ml of Re-suspension
Solution. Vortex to re-suspend the pellet. The bacterial
pellet should be re-suspended completely until no cell
clumps are visible. Let the cell suspension stand at
room temperature for 5 minutes.
3. Add 300 µl of Lysis Solution and mix the contents
gently until the cell suspension is clear. Let the solution
stand for 5 minutes at room temperature. Do not vortex
the solution after adding Lysis Solution.
4. Add 300 µl of Neutralization Solution and
mix the contents by brief vortexing. Let the tube stand
for 5 minutes at room temperature. Transfer the solution
to a microfuge tube and spin at maximum speed for 5
minutes
5. Transfer the supernatant to a microfuge tube and
spin at maximum speed for 5 to 15 minutes.
6. In the mean time, dispense 1.2 ml of DNA Precipitation
Solution into 2 ml of provided microfuge tubes.
7. Add 600 µl of clear supernatant (step 5) into
tube containing DNA Precipitation solution. Vortex the
solution and keep tubes in ice or at –20?C for
10 minutes.
8. Spin tubes for 20 minutes in cold room or use cold
centrifuge.
9. Discard the solution and gently add ice-cold Ethanol
Wash Solution (~1 ml) discard it immediately. In order
to avoid dislodging the pellet, it is recommended to
add ethanol wash from sides of the tube. Note: DNA pellet
may be too small and it may not be visible.
Ethanol Wash Solution:
. Add 1 ml of Conc. DNA Wash solution+ 24 ml water
+ 25 ml Ethanol. Alternatively, add 20 ml of concentrated
10X ethanol wash into a clean bottle; add 180 ml water
and 200 ml of ethanol. Keep bottle containing working
Ethanol Wash Solution at 4?C. Wash solution is stable
for 1 year.
10. Spin tubes for 5 minutes and remove any traces
of ethanol wash with a pipette tip.
11. Add 100 µl of water or TE. Keep tubes at
37 to 42 degree C water bath for about 10 minutes. Vortex
tubes briefly to dissolve the DNA.
Mini Preps:
Scale down the solutions according to growth
media. Protocol is same as above except minor changes
as below:
1. Avoid washing step (step # 9): after spinning the
tube (step # 8), discard solution and spin the tube
again for about 5 minutes in cold room or use cold centrifuge.
Remove any traces of solution.
2. Add ~50 µl of water or TE. Keep tubes at 37
to 42 degree C water bath for about 5 minutes. Vortex
tubes briefly to dissolve the DNA.
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