Key to the kit is our proprietary DNA binding system that allows the highly efficient binding of DNA to our ezBindTM matrix while proteins and other contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or Elution Buffer. The purified DNA is ready for downstream applications such as cloning/subcloning, RFLP, sequencing, and transfection of HEK293 cells.
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-PD1213-01
BMG-PD1213-2
Preps
50
100
Mini Column II
50
100
Buffer A1
25 mL
60 mL
Buffer B1
25 mL
60 mL
Buffer N1*
30 mL
75 mL
Buffer KB*
15 mL
75 mL
DNA Wash Buffer**
12 mL
50 mL
Elution Buffer
15 mL
40 mL
RNase A
110 µL
200 µL
**Buffer N1 and Buffer KB contain chaotropic salts, wear gloves and protective eyewear when handling.
**Add 48 mL (D1213-02) or 200 mL (D1213C) 96-100% ethanol to DNA Wash Buffer before use.
Important:
This kit is designed to purify up to 100 µg plasmid DNA from 6-12 mL culture (See protocol on Page 4). This kit can also be used to purify plasmid DNA from 1-5 mL culture just like regular miniprep (See protocol on Page 8).
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 dissolve at 37°C before use. Keep the cap tightly closed for buffer B1 after use.
Carry out all centrifugations at room temperature.
Buffer N1 and Buffer KB contain a chaotropic salt, wear gloves and protective eyewear when handling.
Materials required but not supplied
96- 100% ethanol.
1.5 mL, 2.0 microcentrifuge tubes.
15 mL conical tubes.
High speed microcentrifuge.
ddH2O.
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 2-3 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, 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 extra wash step by Buffer KB.
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.
