Description : Introduction 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 impurities are removed by Wash Buffer. Nucleic acids are easily eluted with sterile water or Elution Buffer. Unlike other kits in the markets, no chaotropic salts are contained in the buffer of our patented plasmid purification kit. The purified DNA is guanidine/anion exchange resin residues free. Plasmid isolated with traditional protocol normally contains high level of endotoxins (Lipopolysaccharides or LPS). For transfection of endotoxin sensitive cell lines or microinjection, the endotoxins should be removed before the applications. The EZgeneTM endofree system uses a specially formulated buffer that extracts the endotoxin from the plasmid DNA. Two rounds of extraction will reduce the endotoxin level to 0.1 EU (Endotoxin) per µg of plasmid DNA. This kit is designed for fast and efficient purification of plasmid DNA from 15 to 50 mL of E. coli culture. The midi column has a plasmid DNA binding capacity of 250 µg. The purified endofree DNA is ready for high performance of downstream applications such as transfection of endotoxin-sensitive cell lines, primary cultured cells or microinjection. 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. Please contact our customer service for further information and reference Table 1 for the commonly used plasmids, Table 1 Commonly used plasmid and expected yield. Plasmid Origin Copy Numbers Expected Yield (µg per 50 mL) pSC101 pSC101 5 5 pACYC P15A 10-12 5-10 pSuperCos pMB1 10-20 10-20 pBR322 pMB1 15-20 10-20 pGEMR Muted pMB1 300-400 100-150 pBluescriptR ColE1 300-500 100-200 pUC Muted pMB1 500-700 150-250 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 PD1712. 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 mediums 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 midi I column has an optimal biomass of 100-200. For example, if the OD600 is 3.0, the optimal culture volume should be 15 to 50 mL. Culture Volume: Use a flask or tube 4 times bigger in volume than 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 (22-25°C). The Guaranteed shelf life is 12 months from the date of purchase. Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each step. Important Materials supplied by users Kit Contents Catalog # PD2468-00 PD2468-01 PD2468-02 Preps 2 10 25 ezBindTM Columns 2 10 25 Filter Syringe 2 10 25 2.0 ml Microfuge tube 4 20 25 Plastic wrench 1 1 1 Buffer A1 30 mL 70 mL Buffer B1 6 mL 30 mL 70 mL Buffer N3 3 mL 15 mL 30 mL Buffer RET 12 mL 60 mL 135 mL DNA Washing Buffer* 5 mL 24 mL 54mL RNase A (20 mg/mL) 0.6 mg (30 µL) 3 mg (150 µL) 7 mg (350 µL) Endofree Elution Buffer 4 mL 20 mL 60 mL User Manual 1 1 1 *Add 20 mL (PD2468-00) or 96 mL (PD2468-01) or 216 mL (PD2468-02) 96-100% ethanol to each DNA Wash Buffer bottle before use. Before Starting
- Model: PD2468-02
- Manufactured by: Biomiga