Key to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBindTM matrix while proteins and other impurities are removed by wash buffer. Nucleic acids are easily eluted with Elution Buffer. Our proprietary endotoxin removal buffer, Buffer RET, is designed to remove endotoxin by a single washing step without tedious phase partitioning steps. The purified plasmid DNA, with endotoxin level less than 0.1 EU per µg of DNA, is ready for transfetion of endotoxin sensitive cell lines and microinjections.
Storage and Stability
Buffer A1 should be stored at 4°C once RNase A is added. All other materials can be stored at room temperature. The Guaranteed shelf life is 12 months from the date of purchase.
Kit Contents
Catalog#
BMG-PD1222-00
BMG-PD1222-01
BMG-PD1222-02
Mini Column
4
50
250
Buffer A1
2 mL
25 mL
125 mL
Buffer B1
2 mL
25 mL
125 mL
Buffer N3
400 µL
10 mL
50 mL
Buffer RET
2 mL
25 mL
125 mL
Buffer KB
3 mL
25 mL
125 mL
DNA Wash Buffer
1 mL
12 mL
50 mL
RNase A
10 µL
90 µL
420 µL
Endofree Elution Buffer
500 µL
15 mL
50 mL
Important:
RNase A: Spin down RNase A vial briefly. Add the RNase A solution to buffer A1 and mix well before use.
Buffer B1 precipitates below room temperature, it is critical to warm up the buffer at 50°C to dissolve the precipitates before use.
Keep the cap tightly closed for Buffer B1 after use.
Add 48 mL (1222-01) or 200 mL (1222-02) ethanol to DNA Wash Buffer before use.
Ensure the availability of centrifuge capable of 13,000 rpm.
Carry out all centrifugations at room temperature.
Materials required but not supplied
100% ethanol.
1.5 mL microtubes.
Midi/maxiprep: high speed centrifuge, 30 mL high speed centrifuge tubes, 15 /50 ml tubes, and or vacuum manifold capable of generating 200-300 mbar.
Megaprep: Vaccum system, 500 mL bottle (Corning# 430282) or 1,000 mL bottle (#430518) or equivalent.
Important Notes
Copy numbers: The yield of plasmid DNA depends on the origin of the replication and the size of the plasmid. The protocols are optimized for high copy number plasmid purification. For low copy number plasmids, both the culture volume and the buffer volume need to be scaled up 3 to 5 times. Reference the table below for the commonly used plasmids
Plasmid
Origin
Copy Numbers
Expected Yield
(μg / 1 mL)
pSC101
pSC101
5
0.1-0.2
pACYC
P15A
10-12
0.4-0.6
pSuperCos
pMB1
10-20
0.4-1
pBR322
pMB1
15-20
0.6-1
pGEMR
Muted pMB1
300-400
6-7
pBluescriptR
ColE1
300-500
6-8
pUC
Muted pMB1
500-700
8-12
Host Strains: The strains used for propagating plasmid have significant influence on yield. Host strains such as Top 10, DH5a, and C600 yield high-quality plasmid DNA. endA+ strains such as JM101, JM110, HB101, TG1 and their derivatives, normally have low plasmid yield due to either endogenous endonucleases or high carbohydrates released during lysis. We recommend transform plasmid to an endA- strain if the yield is not satisfactory.
Optimal cell mass (OD600 x mL of Culture): This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) for 12 -16 hours to a density of OD600 2.0 to 3.0. If rich medium such as TB or 2xYT are used, make sure the cell density doesn’t exceed 3.0 (OD600). A high ratio of biomass over lysis buffers result in low DNA yield and purity. The maxi column has an optimal biomass of 400-500. For example, if the OD600 is 2.5, the optimal culture volume should be 150 to 200 mL.
