Miniprep

2015-01-13

azim58 - Miniprep

-Official Handbook
--http://www.qiagen.com/literature/render.aspx?id=369
--http://www.qiagen.com/resources/Download.aspx?id={04DD00DF-952E-47AF-8634
-534F108210D8}&lang=en&ver=1
-other protocol

--http://openwetware.org/wiki/Miniprep/Qiagen_kit

Protocol: Plasmid DNA Purification Using the QIAprep
Spin Miniprep Kit and a Microcentrifuge

This protocol is designed for purification of up to 20 µg of high-copy
plasmid DNA from 1–5 ml overnight cultures of E. coli in LB
(Luria-Bertani) medium. For purification of low-copy plasmids and
cosmids, large plasmids (>10 kb), and DNA prepared usingother methods,
refer to the recommendations on page 44.

Note: All protocol steps should be carried out at room temperature.

final concentration of RNase A in Buffer P1: 100 ug/mL
-example calculation: 10000*ug/mL*y/(70*mL)=100*ug/mL -> y = 0.7 mL

Procedure
-Pellet culture >=6,800Xg for 3 min.
--Note that 14 mL polystyrene tubes do not break at 7,000Xg.

-Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to
a microcentrifuge tube.
--Ensure that RNase A has been added to Buffer P1. No cell clumps should
be visible after resuspension of the pellet.
If LyseBlue reagent has been added to Buffer P1, vigorously shake the
buffer bottle to ensure LyseBlue particles are completely dissolved. The
bacteria should be resuspended completely by vortexing or pipetting up
and down until no cell clumps remain.

-Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6
times.
--Mix gently by inverting the tube. Do not vortex, as this will result in
shearing of
genomic DNA. If necessary, continue inverting the tube until the solution
becomes
viscous and slightly clear. Do not allow the lysis reaction to proceed
for more than
5 min.
If LyseBlue has been added to Buffer P1 the cell suspension will turn
blue after addition
of Buffer P2. Mixing should result in a homogeneously colored suspension.
If
the suspension contains localized colorless regions or if brownish cell
clumps are
still visible, continue mixing the solution until a homogeneously colored
suspension
is achieved.

-Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting
the tube
4–6 times.
--To avoid localized precipitation, mix the solution thoroughly,
immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5
ml) may require inverting up to 10 times. The solution should become
cloudy.
If LyseBlue reagent has been used, the suspension should be mixed until
all trace of blue has gone and the suspension is colorless. A homogeneous
colorless suspension indicates that the SDS has been effectively
precipitated.

-Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
microcentrifuge.
--A compact white pellet will form.

-Apply the supernatants from step 4 to the QIAprep spin column by
decanting or
pipetting.

-Centrifuge for 30–60 s. Discard the flow-through.

-Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and
centrifuging for 30–60 s. Discard the flow-through.
--This step is necessary to remove trace nuclease activity when using
endA+ strains such as the JM series, HB101 and its derivatives, or any
wild-type strain, which have high levels of nuclease activity or high
carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not
require this additional wash step.

-Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for
30–60 s.

-Discard the flow-through, and centrifuge for an additional 1 min (2 min
is actually better) to remove residual wash buffer.
--Important: Residual wash buffer will not be completely removed unless
the flow-through is discarded before this additional centrifugation.
Residual ethanol from Buffer PE may inhibit subsequent enzymatic
reactions.
--May want to tap bottom of the QIAprep column on kim wipe to remove any
residual liquid remaining before proceeding to the next step to place the
column into a clean 1.5 mL tube.

-Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To
elute DNA, add 50 μl (we usually do 30 uL for a higher concentration)
Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep
spin column, let stand for 1-5 min, and centrifuge for 1 min.
--May want to vortex and spin the sample before nanodropping to determine
the concentration of DNA.

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