Kit manual

Contents

B A C, PAC , P1 Is ol at i on Pr ot oc ol . . . . . . . . . . . . . . 4 Limited Use and Warranty…………………………………………. 8 Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit Introduction
The EZgeneTM high-capacity-plasmid DNA Isolation Kit is designed for rapid purification of BAC, PAC, cosmid and P1 from bacterial cultures. It is based on a modified alkaline lysis procedure that is specially adapted for spin column. The procedure associated with this kit has been tested using a variety of low copy cosmid, BAC, PAC and P1 in different E. coli strains. In addition, this kit can also be used for high copy plasmid isolation. The expected yield from 200 mL of 2 x
Storage and Stability
All EZgeneTM BAC/PAC DNA Isolation Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows: Buffer A1/RNase A mixture at 4°C, and all other materials at 22-25°C.

Before Starting
Important
 Add whole vial of RNase A to Buffer X1 and Store at 4°C.
 Dilute DNA Wash Buffer with absolute ethanol as follows
PD1314-00 Add 20 mL ethanol to each bottle
PD1314-01 Add 100 mL ethanol to each bottle
PD1314-02 Add 240 mL ethanol to each bottle
 It’s strongly recommended to use 2 x YT media for the cultivation of cosmids,  Dilute BAC Binding Buffer with isopropanol
PD1314-00 Add 24 mL isopropanol to each bottle
PD1314-01 Add 120 mL isopropanol to each bottle
PD1314-02 Add 280 mL isopropanol to each bottle
 Buffer X2 should be kept at room temperature. Check for SDS precipitation
before use. If necessary re-dissolve SDS precipitate by warming. Close the Buffer X2 bottle immediately after use to avoid acidification that may result  Chill Buffer X3 on ice.  Prewarm ddH2O or Elution Buffer at 65°C before elution Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit Kit Contents
Catalog #
PD1314-00
PD1314-01
PD1314-02
* Binding Buffer: Add 24 mL (PD1314-00) or 100 mL (PD1314-01) or 280 mL **DNA Wash Buffer: Add 24 mL (PD1314-00) or 100 mL (PD1314-01) or 216 mL (PD1314-02) of absolute ethanol before use. Materials Supplied by User
 High speed centrifuge and centrifuge tubes capable of at least 12,000 x g  Sterile Deionized Water  50 mL conical tubes.  96-100% Ethanol  96%-100% Isopropanol  One liter 2 x YT medium: Measure 900 mL ddH2O, add 16 g Tryptone, 10 g Yeast Extract, 5 g NaCl, dissolve and adjust pH to 7.0 with 5 N NaOH, adjust to 1000 mL with ddH2O and autoclave. Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit BAC, PAC, P1 and Plasmid Isolation Protocol
1. Inoculate 100-150 mL 2 xYT containing appropriate antibiotic with 100 µL
fresh starter culture. Grow at 37°C for 16-18 h with vigorous shaking. and
inoculate a starter culture of 100-150 mL 2xYT medium containing the
appropriate selective antibiotic. Incubate for 18-20 hours at 37°C with
vigorous shaking (~ 300 rpm). Use a flask with a volume at least 4 times the
volume of the culture.
Note: The best way to prepare a starter culture: Inoculate a single colony
from a freshly grown selective plate into 1 mL LB medium containing the
appropriate antibiotic and grow at 37°C for 8 hours with vigorous shaking
(~250 rpm) and then use this culture as starter culture to inoculate 100-150 mL
of prewarmed (37°C) 2 xYT.
Note: Do not use glycerol stock directly as inoculum. Streak the glycerol stock
onto a agar plate that contains appropriate antibotics and then pick up a single
colony as described to prepare the starter culture.
Note: Do not use a starter culture that has been stored at 4°C.
2. Pellet the bacteria by centrifugation at 5,000 x g for 10 min at room
temperature. Decant or aspirate medium completely and discard.

Note: Pour off the supernatant and blot the inverted tube on paper towels to
remove residual medium completely.
3. Resuspend the bacterial pellet by adding 12 mL Buffer X1/RNase A
solution, resuspend by vortexing or pipetting. Complete resuspension of cell
pellet is vital for obtaining good yields.

4. Add 12 mL Buffer X2 and mix gently but thoroughly by inverting 10 times
to obtain a clear lysate. Incubate at room temperature for 5 min. Avoid
vigorous mixing as this will shear chromosomal DNA and lower plasmid
purity. Do not incubate more than 5 min.

5. Add 12 mL Buffer X3 (prechilled on ice) and gently but thoroughly mix
the sample by inverting 15 times and sharp hand shaking for 2 times. A
flocculent white precipitate forms. Incubate on ice for 5 min.
Note: It is critical to mix the solution well, if the mixture still appears
conglobated, brownish or viscous; more mix is required to completely
neutralize the solution.
Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit 6. Centrifuge at 10,000 x g for 10 min at 4°C. Promptly proceed to the next

7. Carefully transfer the clear supernatant to a new 50 mL conical tube. Add
12 mL Binding Buffer. Mix well by sharp hand shaking for 3 times.

8. Transfer 20 mL of the sample to a DNA column with the collection tube.
Centrifuge at 2500 -5000 x g for 1 min at room temperature. Discard the
flow-through. Transfer the remaining sample to the column and
centrifuge at 2500-5000 x g for 2 min at room temperature. Discard the
flow-through.
Note: When transferring the lysate, some precipitates may float to the top of
the lysate. Carefully transfer the clear lysate by inserting the pipet to lysate to
avoid the precipitates.

9. Add 5 mL Buffer KB，Centrifuge at 2500 -5000 x g for 2 min at room
temperature. Discard the flow-through.

10. Add 10 mL DNA Wash Buffer and centrifuge at 2500 -5000 x g for 2 min
at room temperature. Discard the flow-through.

11. Place the DNA column back into the collection tube and centrifuge at
maximum speed, with the lid open, for 10 min to remove residual ethanol.
Note: This step removes residual ethanol that may otherwise affect DNA yield
and other downstream applications.
Note: As a optional step, bake the column at 65°C for 10 min before adding
Elution Buffer.

12. Transfer the DNA column into a clean 50 mL conical tube, add 0.5 -1 mL
pre-wartmed (65°C) Elution Buffer or ddH2O onto the center of the
membrane. Incubate 5 min at 65°C.

13. Centrifuge at 2500 -5000 x g for 5 min to elute the DNA. Reload the eluted
DNA solution to the column for a second elution will yield another 20% of
the DNA.
Note: Pre-warm Elution Buffer or ddH20 at 65°C and incubate the column at
65°C for 5 min after adding elution buffer or ddH20 will increase the DNA
yield.
Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit Note: The first elution normally yields 60-70% of the DNA bound.
DNA concentration = Absorbance 260 nm x 50 x dilution factor (µg/mL).
The expected yield from 200 mL 2xYT culture is around 30 to 80 µg.
Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit Trouble Shooting Guide
Possible Reason
Solutions
Only use YT medium containing ampicillin. Do not use more than 200 mL culture with the basic protocol. Cells may not have been dispersed adequately prior to the addition of Buffer X2. Make sure to vortex cell Low DNA Yield
Continue inverting vials after adding Buffer X2 to obtain a clear lysate. If not tightly closed, Buffer X2 may need to be replaced. Prepare as follows: 0.2 M NaOH, 1% SDS. Steak out a plate from glycerol stock and avoid repeated freezing/thawing cycles of clones. Always make enough replica plates and use fresh cultures for inoculation. Any remaining cultures can be used to set up fresh glycerol stocks. Check the stock of buffers and age of the buffers. Make sure that the correct volume of buffer has been added to No DNA Eluted
Warm up the Buffer X2 to dissolve the precipitate. Pelleted cells should be completely resuspended with Cells are not completely Buffer X1. Do not add Buffer X2 until an even cell Do not vortex or mix aggressively after adding Buffer X2. High molecular
weight DNA
contamination of
product.
Overgrown culture contains lysed cells and degraded DNA. Do not grow cell for longer than 22 hours. DNA degraded
after the storage
Add 1 vial of RNase to each bottle of Buffer X1. RNA visible on
agarose gel
Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit Limited Use and Warranty
This product is intended for in vitro research use only. Not for use in human.
This product is warranted to perform as described in its labeling and in Biomiga’s
literature when used in accordance with instructions. No other warranties of any
kind, express or implied, including, without limitation, implied warranties of
merchantability or fitness for a particular purpose, are provided by Biomiga.
Biomiga’s sole obligation and purchaser’s exclusive remedy for breach of this
warranty shall be, at the option of Biomiga, to replace the products, Biomiga shall
have no liability for any direct, indirect, consequential, or incidental damage arising
out of the use, the results of use, or the inability to use it product.

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Biomiga EZgeneTM BAC/PAC Maxiprep Isolation Kit

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