Kit Manual
Contents
1. 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 to 3 times Reference Table 1 for the commonly used plasmids Table 1 Commonly used plasmids Plasmid Origin Copy Numbers Expected Yield ug per 200 mL PSC101 pSC101 5 12 pACYC P15A 10 12 25 40 pSuperCos pMBI 10 20 30 50 PBR322 pMBI 15 20 35 50 Page 2 Biomiga EZgene Plasmid ezFilter Maxiprep Kit pGEM Muted pMB1 300 400 350 450 pBluescript CoIE1 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 DH5a DHI DH21 JM106 JM109 SK2262. Add 9 2 mL Buffer C1 and 12 mL 100 ethanol Mix immediately by sharp shaking The mixture of ethanol lysate needs to be centrifuged through the DNA column immediately Immediately apply 20 mL of the lysate ethanol mixture to a DNA column with the collection tube Centrifuge at gt 2 500 x g for 1 minute at room temperature Discard the flow through liquid and put the column back to the collection tube Add the remaining lysate ethanol mixture to the DNA column and centrifuge at 5 000 x g for 1 minutes Discard the flow through liquid and put the column back to the collection tube Add 10 mL 70 ethanol into the column centrifuge at gt 2 500 x g for 1 minute Remove the column from the tube and discard the flow through Biomiga EZgene Plasmid ezFilter Maxiprep Kit Page 7 10 11 12 Reinsert the column into the collection tube Repeat step 9 Centrifuge the column with the lid open at gt 2 500 x g for 10 minutes to remove the ethanol residues Note It is critical to removes ethanol residues completely The remaining ethanol will inhibit the elution of DNA from the column Carefully transfer the column into a sterile clean 50 mL tube and add 1 1 5 mL Sterile ddH O or Elution Buffer to the center of the column and incubate for 1 minute at room temperature Elute the DNA by centrifugation at 5 000 x g for 5 minutes For higher yield reload the eluate in the 50 mL tube to the center of the column and incub
3. slightly clear Incubate at room temperature for 5 minutes to obtain a slightly clear lysate Note Do not incubate longer than 5 minutes Over incubating causes genomic DNA contamination and plasmid damage Page 6 Biomiga EZgene Plasmid ezFilter Maxiprep Kit Add 2 8 mL Buffer C1 mix immediately by inverting shaking the vial for 5 times and sharp hand shaking for 5 times Note Adding ice cold C1 or incubating the lysate in ice will decrease the floating precipitates at step 6 Optional 1 Transfer the lysate to a high speed centrifuge tube and centrifuge at 14 000 x g for 10 minutes at room temperature Carefully transfer the clear supernatant into a 15 mL tube avoid the floating precipitates Note If the rotor is cold incubate the lysate at room temperature for 10 minutes and then perform centrifugation as described Optional 2 Pour the lysate directly into the barrel of the filter syringe Insert the syringe to a clean 50 mL tube not supplied set in a rack Allow the cell lysate to sit for 10 minutes The white precipitates should float to the top Hold the filter syringe barrel over the 15 mL tube and gently insert the plunger to expel the cleared lysate to the tube stop when feel major resistance some of the lysate may remain in the flocculent precipitate do not force the residual lysate through the filter Carefully transfer the clear supernatant into a 50 mL tube avoid the floating precipitates
4. 7 SRB XLO TOP10 DHIOB JM103 JM107 SK1590 MM294 Stb 2 XLI Blue BJ5182 DH20 JM105 JM108 SK1592 Select96 Stbl4 e EndA Strains of E Coli C600 JM110 RRI ABLE C CJ236 KW251 P2392 BL21 DE3 HB101 TG TB1 ABLE K DHDS BL21 DE3 LE392 PR700 JM101 JM83 TKB1 HMSI74 ES1301 M1061 Q358 BMH 71 18 pLysS All NM strains All Y strains Optimal Cell Mass OD o 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 o9 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 ODg o9 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 ODgo9 is 2 5 the optimal culture volume should be 200 mL Biomiga EZgene Plasmid ezFilter Maxiprep Kit Page 3 Culture Volume Use a flask or tube 4 times larger 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 Al 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
5. Contents ond P M 1 TNO CUCU ON eM E 2 Important Notes ore en t eese dor EH un ake 2 Storage and Stability oiii o Het t id ep SERE de ass 4 Before Starting iiic eS IHE XR Hn OE ea T ESAN EEEE 4 ImpoOtrtatit 15 2 B ao en e eiie cun ne tele 4 Materials supplied by users seen 5 Kit Contents b ERU Hab ioi die ee ete 5 Safety Information uei rn irre ER SENSUS Se ii n iiias 5 EZgene Plasmid Maxiprep Spin Protocol seeee 6 Purification of Low Copy Number Plasmid andCosmid 9 Trouble Shooting Cyulde arie ater dee dede duos Gace ee 10 Biomiga EZgene Plasmid ezFilter Maxiprep Kit Page 1 Introduction Key to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezBind 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 This kit is designed for fast and efficient purification of plasmid DNA from 100 to 250 mL of E coli culture The maxi column has a plasmid DNA binding capacity of 1 mg The purified plasmid DNA is ready for downstream applications such as transfection of robust cells such as HEK293 restriction mapping library screening
6. arose gel DNA doesn t freeze or smell of ethanol Ethanol traces not completely removed from column Make sure that no ethanol residual remaining in the silicon membrane before elute the plasmid DNA Re centrifuge or vacuum again if necessary Biomiga EZgene Plasmid ezFilter Maxiprep Kit Page 11
7. ate for 1 minute at room temperature Elute the DNA again by centrifugation at 5 000 x g for 5 minutes Note If ddH5O is used for eluting DNA make sure the pH is gt 7 0 Note The DNA is ready for downstream applications such as cloning subcloning RFLP Library screening in vitro translation sequencing transfection of HEK293 cells Note It s highly recommended to remove the endotoxin PD1514 if the DNA is used for transfection of endotoxin sensitive cell lines primary cultured cells or microinjection Note Two elutions give rise to maximum DNA yield For maximum yield and higher concentration pool the elutions together add 0 1 volume 3M KAc or NaAc pH 5 2 and 0 7 volume isopropanol Mix well and aliquot the sample to 2 0 mL microtubes Centrifuge at top speed for 10 min Remove the supernatant Wash the DNA with 800 uL 70 ethanol centrifuge for 5 min carefully decant Air dry the pellet for 10 min Resuspend the DNA in Elution Buffer or sterile ddH5O Page 8 Biomiga EZgene Plasmid ezFilter Maxiprep Kit The DNA concentration can be determined by a spectrophotometer DNA concentration ug mL OD x 50 x dilution factor Purification of Low Copy Number Plasmid andCosmid The yield of low copy number plasmid is normally around 0 1 1 ug mL of overnight culture For isolating low copy number or medium copy number plasmid DNA use the following guideline 1 Culture volume Use 2 x volumes of the high copy nu
8. mber culture Use 400 mL for the maxiprep 2 Use2 x volumes of the Buffer A1 Buffer B1 and Buffer C1 and 100 ethanol Additional buffers can be purchased from Biomiga 3 Use same volume of Wash Buffer 70 ethanol and Elution Buffer Biomiga EZgene Plasmid ezFilter Maxiprep Kit Page 9 Trouble Shooting Guide Problems Possible Reasons Suggested Improvements Low Yield Poor Cell lysis e Resuspend pellet thoroughly by votexing and pipetting prior adding Buffer B1 e Make fresh Buffer B1 if the cap had not been closed tightly Buffer B1 0 2N NaOH and 1 SDS Low Yield Bacterial culture Grow bacterial 12 16 hours Spin overgrown or not down cultures and store the pellet at fresh 20 C if the culture is not purified the same day Do not store culture at 4 C over night Low Yield Low copy number Increase culture volume Increase plasmid the volume of Buffer Al B1 Cl and ethanol proportionally with the ratio of 1 1 1 2 1 2 No DNA Plasmid lost in Host Prepare fresh culture E coli Page 10 Biomiga EZgene Plasmid ezFilter Maxiprep Kit Genomic DNA Over time incubation Do not vortex or mix aggressively contamination after adding Buffer after adding Buffer Bl Do not Bl incubate more than 5 minutes after adding Buffer B1 RNA contamination RNase A not added Add RNase A to Buffer Al to Buffer Al Plasmid DNA floats out of wells while running in ag
9. 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 step Important e RNase A It is stable for more than half a year when stored at room temperature Spin down the RNase A vial briefly Add the RNase A solution to Buffer Al and mix well before use and then store at 4 C e Buffer B1 precipitates below room temperature It is critical to warm up the buffer at 50 C to dissolve the precipitates before use e Incubating Buffer C1 at 4 C before experiment will decrease the floating precipitates e Keep the cap tightly closed for Buffer B1 after use e Make sure the availability of centrifuge and vacuum manifold especially after mixing the lysate with ethanol the sample needs to be processed immediately either by centrifugation or vacuum Page 4 Biomiga EZgene Plasmid ezFilter Maxiprep Kit e Carry out all centrifugations at room temperature Materials supplied by users e 70 ethanol and 100 ethanol e High speed centrifuge e 30 mL high speed centrifuge tubes e 50 mL tubes Kit Contents Catalog PD1512 00 PD1512 01 PD1512 02 Preps 2 10 25 ezBind Columns 2 10 25 Ome 2 10 25 Buffer A1 22mL 110 mL 270 mL Buffer B1 22mL 110 mL 270 mL Buffer C1 27 mL 135 mL 330 mL RNase A 2 2 mg 11 mg 27 mg 20 mg mL 110 uL 550 uL 1 35 uL El
10. ution Buffer 6 mL 30 mL 60 mL User Manual 1 1 1 Safety Information Buffer C1 contains acidic acid wear gloves and protective eyewear when handling Biomiga EZgene Plasmid ezFilter Maxiprep Kit Page 5 EZgene Plasmid Maxiprep Spin Protocol 1 Inoculate 150 200 mL LB containing appropriate antibiotic with 100 uL fresh starter culture Incubate at 37 C for 14 16 h with vigorous shaking Note The best way to prepare a starter culture Inoculate a single colony from a freshly grown selective plate into 1 ml LB medium containing the appropriate antibiotic and grow at 37 C for 6 8 h with vigorous shaking 250 rpm Note Do not use more than 200 mL culture or cell mass greater than 550 The buffer volume needs to be scaled up if processing over 200 mL of culture Note Do not use a starter culture that has been stored at 4 C Note Do not grow starter culture directly from glycerol stock 2 Harvest the bacterial by centrifugation at 5 000 x g for 10 minutes at room temperature Pour off the supernatant and blot the inverted tube on paper towels to remove residual medium completely 3 Add 10 mL Buffer A1 Add RNase A to Buffer A1 before use and completely resuspend bacterial pellet by vortexing or pipetting Complete resuspension is critical for optimal yields 4 Add 10 mL Buffer B1 mix gently but thoroughly by inverting 5 10 times If necessary continue inverting the tube until the solution becomes
Download Pdf Manuals
Related Search