Protocol for PlasmidMAX™ DNA Isolation Kit

PlasmidMAX DNA Isolation Kit
Cat. No. PMX51050
The PlasmidMAX DNA Isolation Kit provides all of the reagents necessary to purify
plasmids free of RNA and genomic DNA. The resulting high quality DNA permits
sequencing reads of at least 500 bases. This simple procedure requires only two 1.5 ml
tubes, thus avoiding excess plastic-ware waste. The purification method is scalable for
larger volumes, which allows the customer to avoid purchasing separate kits for varying
sample sizes. No spin columns or other specialized equipment is required.
Product Specifications:
Storage: Store the RiboShredder RNase Blend at –20°C in a freezer without a defrost
cycle. Store the remainder of the kit components at room temperature.
Storage Buffer: RiboShredder RNase Blend is supplied in a 50% glycerol solution
containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1 mM EDTA.
Quality Control: The PlasmidMAX DNA Isolation Kit is function-tested by purifying
plasmid DNA from E. coli. DNA quality and yield are assayed by restriction enzyme
digestion, gel electrophoresis, spectrophotometry and fluorimetry.
Related Products: The following products are also available:
− FosmidMAX DNA Purification Kit
− BACMAX DNA Purification Kit
Desc.
Quantity
PlasmidMAX DNA Isolation Kit Contents
The PlasmidMAX DNA Isolation Kit contains sufficient reagents to perform 50 extractions. The kit
contains the following reagents:
PlasmidMAX Solution 1
10 ml
PlasmidMAX Solution 2
20 ml
PlasmidMAX Solution 3
15 ml
RiboShredder RNase Blend
25 μl
RiboShredder Buffer
2.5 ml
Plasmid Precipitation Solution
1.3 ml
TE Buffer
1.3 ml
(10 mM Tris-HCl [pH 7.5], 1 mM EDTA)
www.epicentre.com Lit. # 232 • 6/2011 1

PlasmidMAX DNA Isolation Kit
Plasmid DNA Purification Protocol
1. Pellet 1-1.5 ml aliquots of late log or stationary phase E. coli cultures for 1 minute in a
microcentrifuge at maximum speed.
2. Remove the supernatant with a pipet tip.
3. Thoroughly resuspend the bacterial pellet in 200 μl of PlasmidMAX Solution 1 by
pipetting the mixture up and down.
4. Add 400 μl of PlasmidMAX Solution 2
Note: If Solution 2 has precipitated, warm briefly at 37°C to redissolve.)
Mix the contents by inverting the tube 4-6 times until the cell suspension “clears”.
5. Add 300 μl of PlasmidMAX Solution 3. Mix the contents by inverting the tube 4-6
times. A white precipitate consisting of cellular debris and chromosomal DNA will
appear.
6. Pellet cellular debris by spinning in a microcentrifuge at maximum speed for 10
minutes at 4°C. Transfer the supernatant solution to a clean tube. Be careful not to
take any white precipitate to the next tube.
7. Add 550 μl of 100% isopropanol. Mix the contents by inverting the tube and spin the
tube in a microcentrifuge at maximum speed for 10 minutes.
8. Completely remove the isopropanol with a pipet tip.
Respin the tube for 10 seconds and further remove any residual isopropanol.
9. Add 1 μl of RiboShredder RNase Blend to 100 μl of RiboShredder Buffer.
Mix and add 50 μl of this solution to each DNA pellet (100 μl is enough for 2 pellets.).
Mix thoroughly by vortexing.
10. Incubate at 37°C for 15 minutes.
11. Add 25 μl of Plasmid Precipitation Solution.
12. If the plasmid is >10-15 kb, invert the tube 4-6 times. Otherwise, briefly vortex and
then let the sample sit at room temperature for 10 minutes.
13. Pellet the plasmid DNA in a microcentrifuge at maximum speed for 10 minutes at
room temperature or 4°C.
14. Carefully remove the supernatant solution. Rinse the pellet with 200 μl of 70%
ethanol being careful not to lose the plasmid DNA.
15. Spin in a microcentrifuge at maximum speed for 10 minutes at room temperature or
4°C. Carefully remove the supernatant fluid.
16. Resuspend the pellet (which may be invisible) in 25 μl of TE Buffer.
17. Measure the DNA concentration with fluorimetry and Hoechst Dye 33258 or by
absorbance at 260 nm.
BACMAX, FosmidMAX, PlasmidMAX, and RiboShredder are trademarks of Epicentre, Madison, Wisconsin.
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Notes
techhelp@epicentre.com • (800) 284-8474 3

726 Post Road, Madison, WI 53713 (800) 284-8474 (608) 258-3080 Fax (608) 258-3088
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