Description : Introduction Key to the plasmid purification kit is our proprietary DNA binding system that allows the high 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, our patented plasmid purification kit has no chaotropic salts in the buffer. The purified DNA is guanidine/anion exchange resin residues free. The EZgeneTM endofree system uses a specially formulated buffer that extracts the endotoxin from the bacterial lysate. The endotoxin level is less than 0.1 EU (Endotoxin) per µg of plasmid DNA. This kit is designed for fast and efficient purification of plasmid DNA from 150 to 400 mL of E. coli culture. The maxi column has a DNA binding capacity of 1200 µg. The purified endofree DNA is ready for downstream applications such as transfection of endotoxin-sensitive cell lines, primary cultured cells or microinjection. Two endotoxin removal procedures are provided. Protocol A removes endotoxin during the purification of plasmid DNA and Protocol B removes endotoxin after the purification of plasmid DNA. 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. Reference Table 1 for the commonly used plasmids, Table 1 Commomly used plasmids Plasmid Origin Copy Numbers Expected Yield (µg per 200 mL) pSC101 pSC101 5 12 PACYC P15A 10-12 25-40 pSuperCos pMB1 10-20 30-50 pBR322 pMB1 15-20 35-50 pGEMR Muted pMB1 300-400 350-450 pBluescriptR ColE1 300-500 450-600 PUC Muted pMB1 500-700 700-1,000 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 PD1713. 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. The maxi column has an optimal biomass of 450-550. For example, if the OD600 is 2.5, the optimal culture volume should be 200 mL. 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 and Buffer ER should be stored at 4°C. All other materials can be stored at room temperature (22-25°C). The Guaranteed shelf life is 12 months from the date of purchase. Kit Contents Catalog # PD1522-00 PD1522-01 PD1522-02 Preps 2 10 25 ezBindTM Columns 2 10 25 Filter syringe (60 mL) 2 10 25 Buffer A1 22 mL 110 mL 270 mL Buffer ER 1.2 mL 5.5 mL 13 mL Buffer B1 22 mL 110 mL 270 mL Buffer D1 2.5 mL 11 mL 27 mL Buffer N3 8 mL 40 mL 80 mL Buffer RET* 40 mL 200 mL 2×250 mL RNase A (20 mg/mL) 2.2 mg (110 μL) 11 mg (550 μL) 27 mg (1.35 mL) Endofree Elution Buffer 12 mL 60 mL 135 mL User Manual 1 1 1 Safety Information Buffer N3, RET contain acetic acid, wear gloves and protective eyewear when handling. *Buffer ER and RET are for endotoxin removal. Before Starting Alternative endotoxin removal procedures are provided. Protocol A removes endotoxin during the purification of plasmid DNA and Protocol B removes endotoxin after the purification of plasmid DNA. Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each step. Operating Protocol Important
Materials supplied by users
- Model: PD1522-01
- Manufactured by: Biomiga