Plasmid Midi Kit I (2 Preps)
Cat. No.: BMG-PD1411-00
Size: 2 Preps
Description
Key to the kit is our proprietary DNA binding systems that allow 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 TE buffer. Unlike all other rivals, Biomiga’s patented plasmid purification kit has no guanidine salt in the buffer, the purified DNA is guanidine/ion exchange resin residues free which enable the high performance of downstream applications such as transfection, restriction mapping, library screening, sequencing, as well as gene therapy and genetic vaccinations.
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-PD1411-01 |
BMG-PD1411-2 |
| Preps |
10 |
25 |
| Midi Column |
10 |
25 |
| Buffer A1 |
30 mL |
70 mL |
| Buffer B1 |
30 mL |
70 mL |
| Buffer C1* |
35 mL |
90 mL |
| DNA Wash Buffer** |
50 mL |
50 mL |
| RNase A |
3 mg |
7 mg |
| Elution Buffer |
10 mL |
30 mL |
*Buffer C1 contains acetic acid, wear gloves and protective eyewear when handling.
**DNA Wash Buffer: Add ethanol before use.
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 37°C to dissolve the precipitates before use.
- DNA Wash Buffer: Add 200 mL 96-100% ethanol to each bottle before use.
- Keep the cap tightly closed for buffer B1 after use.
- Ensure the availability of centrifuge capable of 13,000 rpm.
- Carry out all centrifugations at room temperature.
Materials required but not supplied
- 70% ethanol and 100% ethanol.
- High speed centrifuge, 30 mL high speed centrifuge tubes, 15 ml conical 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 |
High copy |
Low copy |
| pACYC |
P15A |
|
10-12 |
| pSC101 |
pSC101 |
|
5 |
| pSuperCos |
pMB1 |
|
10-20 |
| pBR322 |
pMB1 |
|
15-20 |
| pUC |
Muted pMB1 |
500-700 |
|
| pGEMR |
Muted pMB1 |
300-400 |
|
| pBluescriptR |
ColE1 |
300-500 |
|
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 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 PD1712.
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 OD
600 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 buffer results in low DNA yield and purity. The midi II column has an optimal biomass of 150-250. For example, if the OD
600 is 3.0, the optimal culture volume should be 50 to 80 mL.
