Description :
Introduction
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 contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or elution buffer.
Unlike other procedures, our 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.
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 times. Reference Table 1 for the commonly used plasmids,
Table 1 Commonly used plasmids.
Plasmid | Origin | Copy Numbers | Expected Yield (µg per 500 mL) |
pSC101 | pSC101 | 5 | 50-60 |
pACYC | P15A | 10-12 | 80-100 |
pSuperCos | pMB1 | 10-20 | 80-150 |
pBR322 | pMB1 | 15-20 | 100-150 |
pGEMR | Muted pMB1 | 300-400 | 2000-2500 |
pBluescriptR | ColE1 | 300-500 | 2000-3000 |
pUC | Muted pMB1 | 500-700 | 3000-4000 |
Host Strains: The strains used for propagating plasmid have significant influence on yield. Host strains such as Top 10 and DH5a 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 (Table 2), we recommend use product PD1714.
Table2 endA strains of E. Coli.
EndA- Strains of E. Coli |
DH5α | DH1 | DH21 | JM106 | JM109 | SK2267 | SRB | XLO |
TOP10 | DH10B | JM103 | JM107 | SK1590 | MM294 | Stbl2TM | XL1-Blue |
BJ5182 | DH20 | JM105 | JM108 | SK1592 | Select96TM | Stbl4TM | XL10-Gold |
EndA+ Strains of E. Coli |
C600 | JM110 | RR1 | ABLE® C | CJ236 | KW251 | P2392 | BL21(DE3) |
HB101 | TG1 | TB1 | ABLE® K | DH12STM | LE392 | PR700 | BL21(DE3) pLysS |
JM101 | JM83 | TKB1 | HMS174 | ES1301 | M1061 | Q358 | BMH 71-18 |
All NM strains | All Y strains |
| | | | | | | | | | | | | | |
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.
Culture Volume: Use a flask or tube with a volume at 4 times the culture medium to secure optimal condition for bacteria growth. Don't exceed the maximum culture volume suggested in the protocol. Incomplete lysis due to over amount of bacterial culture results in lower yield and less purity.
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 (22-25oC). The Guaranteed shelf life is 12 months from the date of purchase.
Before Starting
Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps.
Important:
- RNase A: It is stable for more than half a year when stored at room temperature. Spin down RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well before use.
- Buffer ER should be stored at 4°C.
- Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50°C to dissolve the precipitates before use.
- Buffer N3 may form precipitates below 10°C, warm up at 37°C to dissolve the precipitates before use.
- Keep the cap tightly closed for Buffer B1 after use.
- Make sure the availability of centrifuge and vacuum manifold, especially, after mixing the lysate with ethanol, the sample needs to be processed immediately by vacuum.
Materials supplied by users:
- 70% ethanol and 100% ethanol.
- Pump-driven vacuum system, 1,000 mL bottle (Corning# 430518 or 430282) or equivalent pyrex glass bottles.
- 50 mL conical tubes.
- High speed centrifuge tube for endotoxin removal if desired.
Kit Contents
Catalog# | PD1622-00 | PD1622-01 | PD1622-02 |
Preps | 1 | 2 | 10 |
DNA Unit | 1 | 2 | 10 |
Filter Unit | 1 | 2 | 10 |
Replacement Cup | 1 | 4 | 20 |
Buffer A1 | 60 mL | 130 mL | 2×320 mL |
Buffer ER | 3 mL | 6.5 mL | 32 mL |
Buffer B1 | 60 mL | 130 mL | 2×320 mL |
Buffer D1 | 6 mL | 13 mL | 64 mL |
Buffer N3 | 15 mL | 35 mL | 160 mL |
Buffer RET | 130 mL | 260 mL | 3×420 mL |
RNase A (20 mg/mL) | 6 mg (300 µL) | 12.5 mg (625 µL) | 2×32 mg (2×1.6 mL) |
Endofree Elution Buffer | 30 mL | 60 mL | 270 mL |
User Manual | 1 | 1 | 1 |
Safety Information
- Buffer N3 contains acetic acid, use gloves and protective eyewear when handling.
- Buffer N3, Buffer RET contains chaotropic salts, which may form reactive compounds when combines with bleach. Do not add bleach or acidic solutions directly to the preparation waste.
Operating Protocol