Key to the kit is our proprietary DNA binding system that allows the highly efficient binding of DNA to our matrix while proteins and other contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with Elution buffer. The purified DNA is ready for downstream applications such as cloning/subcloning, RFLP, sequencing, and transfection of HEK293 cells. The yield from 1 mL culture is typical around 8 to 12 ug. The mini column has a DNA binding capacity of 40 ug.
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 18 months from the date of purchase.
Kit Contents
Catalog#
BMG-PD1211-01
BMG-PD1211-02
Preps
50
250
ezBind Columns
50
250
Buffer A1
15 mL
65 mL
Buffer B1
15 mL
65 mL
Buffer N1*
20 mL
90 mL
Buffer KB
15 mL
70 mL
DNA Wash Buffer**
12 mL
50 mL
Elution Buffer
10 mL
30 mL
RNase A
50 µL
210 µL
*Buffer N1 contains chaotropic salts, wear gloves and protective eyewear when handling.
**Add 48 mL (D1211-01) or 200 mL (D1211-02) 96-100% ethanol to DNA Wash Buffer before use.
Important:
RNase A: Spin down RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use.
Add 48 mL (1211-01) or 200 mL (D1211-02) 96-100% ethanol to DNA Wash Buffer before use
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 12,000 rpm.
Carry out all centrifugations at room temperature.
Buffer N1 contains a chaotropic salt, wear gloves and protective eyewear when handling.
Materials required but not supplied
96- 100% ethanol.
1.5 mL microcentrifuge tubes
High speed microcentrifuge.
Important Notes
Plasmid 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 2 to 3 times.
Plasmid
Origin
Copy Numbers
Expected Yield (µg per 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, JM109, JM110, HB101, TG1 and their derivatives, normally have low plasmid yield. We recommend transform plasmid to a endA- strain if the yield is not satisfactory. For endA+ strains, we recommend use Buffer KB to remove the endonuclease before washing step.
Culture Medium: This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) to 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 cell density over lysis buffer results in low DNA yield and purity. For over amount of cell numbers, either reduce the biomass or scale up the volumes of Buffer A1, B1 and N1.
