morgan@go2biotech.comGDP103-TIANprep Mini Plasmid Kit
TIANprep Mini Plasmid Kit——For purification of molecular biology
grade from 1-5 ml overnight bacteria culture
TIANprep Mini Plasmid Kit
(Spin Column)
Cat. no. GDP103
Storage
TIANprep Mini Plasmid Kit can be stored dry at room temperature (15-30°C) for up to 15 months without showing any reduction in performance and quality. If any precipitate forms in the buffers, it should be dissolved by warming the buffers at 37°C before use. RNase A (10 mg/ml) can be stored
for 15 months at room temperature (15-30°C). After addition of RNase A,
Buffer P1 is stable for 6 months at 2-8°C.
Introduction
TIANprep Mini Plasmid Kit is based on alkaline lysis technology followed
by adsorption of DNA onto silica membrane in the presence of high salt.
Plasmid DNA purified with TIANprep Mini Plasmid Kit is immediately ready
for use. Phenol extraction and ethanol precipitation are not required. Highquality plasmid DNA is eluted in a small volume of Tris Buffer or deionized water. This protocol is designed for purification of plasmid DNA from 1-5ml overnight cultures of E. coli in LB (Luria-Bertani) medium.
Plasmid DNA prepared by TIANprep Mini Plasmid Kit is suitable for a
variety of routine applications including restriction enzyme digestion,
sequencing, library screening, ligation and transformation, in vitro
translation, and transfection of culture cells.
Important Notes Before starting
- Add the provided RNase A solution to Buffer P1 before use (use 1 vial
RNase A per bottle Buffer P1), mix, and store at 2-8°C. - Check Buffer BL, P2 and P3 before use for salt precipitation. If necessary,
dissolve the buffer by warming at 37°C for several minutes. - Avoid direct contact of Buffer P2 and P3, immediately close the lid after
use. - All centrifugation steps are carried out at 12,000 rpm (~13,400× g) in a
table-top microcentrifuge at room temperature (15-30°C). - The amount of extracted plasmid is related to cells concentration and
plasmid copy. - Using Buffer BL to treat spin columns could activate silica membrane at
maximum degree and higher yield. - After treated with Buffer BL, use the Spin Column soon, otherwise longterm placement may affect the purifying effect.
- Buffer PD can effectively remove residual protein. This step is essential
when working with endA+strains such as TG1, BL21, HB101, ET12567,
JM series, etc., to ensure that plasmid DNA is not degraded.
Reagents need to be prepared by Customer
96-100% ethanol
Protocol
Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the
adding volume
- Column equilibration: Place a Spin Column CP3 in a clean collection
tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000
rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flowthrough, and put the Spin Column CP3 back into the collection tube.
(Please use freshly treated spin column). - Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation
at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge
for 1 min at room temperature (15-30°C), then remove all traces of
supernatant until all medium has been drained (For large volume of
bacterial cells, please harvest to one tube by several centrifugation
step.) - Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase
A has been added).The bacteria should be resuspended completely by
vortex or pipetting up and down until no cell clumps remain.
Note: No cell clumps should be visible after resuspension of the pellet,
otherwise incomplete lysis will lower yield and purity. - Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the
tube 6-8 times.
Note: 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 the lysate is
still not clear, please reduce bacterial pellet. - Add 350 μl Buffer P3 and mix immediately and gently by inverting the
tube 6-8 times. The solution should become cloudy. Centrifuge for 10
min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.
Note: To avoid localized precipitation, mix the solution thoroughly,
immediately after addition of Buffer P3. If there is still white
precipitation in the supernatant, please centrifuge again. - Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3
in a collection tube) by decanting or pipetting. Centrifuge for 30-60 sec
at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin
Column CP3 back into the Collection Tube. - (Optional) Wash the Spin Column CP3 by adding 500 µl Buffer PD and
centrifuge for 30-60 sec at 12,000 rpm (~13,400 × g). Discard the flowthrough and put Spin Column CP3 back to the collection tube.
This step is recommended to remove trace nuclease activity when using
endA+strains such as TG1, BL21, HB101, ET12567, JM series, etc. end Astrains such as DH5α, TOP10 can skip this step. - Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure that
ethanol (96%-100%) has been added)and centrifuge for 30-60 sec at
12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin
Colum CP3 back into the Collection Tube. - Repeat Step 8.
- Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to
remove residual Buffer PW.
Note: Residual ethanol from Buffer PW may inhibit subsequent
enzymatic reactions. We suggest open CP3 lid and stay at room
temperature for a while to get rid of residual ethanol. - Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To
elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column
CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm
(~13,400 × g).
Note: If the volume of eluted buffer is less than 50 μl, it may affect
recovery efficiency. The pH value of eluted buffer will have some
influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is
suggested to elute plasmid DNA. For long-term storage of DNA,
eluting in Buffer EB and storing at -20°C is recommended, since
DNA stored in water is subject to acid hydrolysis. Repeat step 11 to
increase plasmid recovery efficiency.
Extraction of Low Copy or Large Plasmid (>10 kb)
For low copy plasmids and plasmids larger than 10 kb, the amount of
bacteria should be increased. It is recommended to use 5-10 ml overnight
culture, and the volume of Buffer P1, P2 and P3 should be increased in
proportion. Buffer EB should be preheated in 65-70°C water bath, and the
incubation time for adsorption and elution can be appropriately prolonged
to increase the extraction efficiency. The other steps are the same as the
above protocol.
GDP103-TIANprep Mini Plasmid Kit
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