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-PD1520-00
BMG-PD1520-01
BMG-PD1520-02
Maxi Columns
2
10
25
Buffer A1
30 mL
120 mL
260 mL
Buffer B1
30 mL
120 mL
260 mL
Buffer N3
10 mL
40 mL
80 mL
Buffer RET
30 mL
120 mL
260 mL
DNA Wash Buffer
12 mL
50 mL
2x50 mL
RNase A
110 µL
400 µL
1000 µL
Endofree Elution Buffer
3 mL
15 mL
50 mL
Important:
RNase A: Spin down RNase A vial briefly. Add RNase A to Buffer A1. Mix well before use. Store at 4°C.
Buffer B1 precipitates below room temperature, it is critical to warm up the buffer at 37°C to dissolve the precipitates before use.
Keep the cap tightly closed for buffer B1 after use.
Ensure the availability of centrifuge capable of 5,000 x g.
Carry out all centrifugations at room temperature.
Add 48 mL (PD1520-00) or 50 mL (PD1520-01 and PD1520-02) to each DNA Wash Buffer before use.
Materials required but not supplied
100% ethanol
50 mL conical tubes.
Centrifuge with swing bucket rotor and centrifuge tubes.
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 /200 mL)
pSC101
pSC101
5
10-15
pACYC
P15A
10-12
20-25
pSuperCos
pMB1
10-20
20-40
pBR322
pMB1
15-20
30-40
pGEMR
Muted pMB1
300-400
400-500
pBluescriptR
ColE1
300-500
400-600
pUC
Muted pMB1
500-700
600-1200
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. For purifying plasmid DNA from endA+ strains, we recommend use product number PD1713.
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.
