Kit Manual
Contents
1. 22 25 C The Guaranteed shelf life is 12 months from the date of purchase Biomiga EZgene Plasmid Midiprep Kit Page 3 Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each step Important 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 Store 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 Keep the cap tightly closed for Buffer B1 after use Make sure the availability of centrifuge especially after mixing the lysate with ethanol the sample needs to be processed immediately either by centrifugation Carry out all centrifugations at room temperature Materials supplied by users 70 ethanol and 100 ethanol High speed centrifuge 30 mL high speed centrifuge tubes 15 mL and 50 mL conical tubes 1 5 mL tubes Isopropanol if precipitate the plasmid DNA Page 4 Biomiga EZgene Plasmid Midiprep Kit Kit Contents Catalog PD1411 00 PD1411 01 PD1411 02 Preps 2 10 25 ezBind Columns 2 10 25 Buffer A1 6 mL 30 mL 70 mL Buffer B1 6 mL 30 mL 70 mL Buffer C1 7 mL 35 mL 85 mL Buffer KB 7 mL 35 mL 85 mL RNase A 0 6 mg 3mg 7 mg 20 mg mL 30 uL 152. DNA again by centrifugation at gt 2 500 x g for 5 min Note If ddH5O is applied please make sure the pH is no less than 7 0 7 0 8 5 is preferred NaOH could be used to adjust the pH of ddH5O Note The DNA is ready for downstream applications such as cloning subcloning RFLP Library screening in vitro translation sequencing transfection of robust HEK293 cells It s highly recommended to remove the endotoxin PD1415 if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection Note The first elution normally yield 60 80 of the DNA bound Two elutions increase the DNA recovery up to 90 For maximum yield and higher concentration Biomiga EZgene Plasmid Midiprep Kit Page 7 pool the elutions together add 0 1 volume 3M NaAc and 0 7 volume isopropanol Mix well by sharp hand shaking and 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 5 10 min Resuspend the DNA in Elution Buffer or Sterile ddH5O DNA concentration ug mL OD2 6onm x 50 x dilution factor Page 8 Biomiga EZgene Plasmid Midiprep Kit Purification of Low Copy Number Plasmid Cosmid 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 th
3. from column Make sure that no ethanol residual remaining in the silicon membrane before eluting the plasmid DNA Re centrifuge or vacuum again if necessary Page 10 Biomiga EZgene Plasmid Midiprep Kit
4. 0 uL 350 uL Elution Buffer 5mL 25 mL 60 mL User Manual 1 1 1 Safety Information e Buffer C1 contains acidic acid wear gloves and protective eyewear when handling e Buffer Cl and KB contains chaotropic salts which may form reactive compounds when combines with bleach Do not add bleach or acidic solutions directly to the preparation waste Biomiga EZgene Plasmid Midiprep Kit Page 5 EZgene Plasmid Midiprep Spin Protocol 1 Inoculate 15 50 mL LB containing appropriate antibiotic with 50 uL fresh starter culture Incubate at 37 C for 14 16 hours 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 8 hours with vigorous shaking 250 rpm and then use the culture as starter culture Note Do not use more than 50 mL culture or cell mass greater than 150 The buffer volume needs to be scaled up if processing over 100 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 3 Add 2 5 mL Buffer Al Add RNase A to Buffer Al before use and completely resuspend bacterial pellet by vorte
5. Table of Contents CCOftents 4 cesset iter A E PERSE 1 Introduction yeaa eoo EXE IA AREA Dep Ex tele ea ars 2 Important Not sieer cece eeee eee ne ene I m e me heme 2 Storage and Stability cece cece cee nee e cece ence eens eene 3 Before Starting cease sas ak eee ee es ae ea N uds 4 Kit Contents eet d nee ese eee lade cule s a beat 5 Safety Information ccc cece aA KEA enc eee ene ene EE AE 5 EZgene Plasmid Midiprep Spin Protocol 6 Purification of Low Copy Number Plasmid and Cosmid 9 Trouble Shooting Guide sse 10 Biomiga EZgene Plasmid Midiprep Kit Page 1 Introduction Key to this kit is our proprietary DNA binding systems that allow DNA exclusively and efficiently bind 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 buffers the purified DNA is guanidine anion exchange resin residues free which enable the high performance of downstream applications 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 ug The yield from 50 mL culture is typically around 150 to 250 ug The purified DNA is ready for high performance of downstream applicat
6. e high copy number culture Use 100 mL for midiprep 2 Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer C1 Additional buffers can be purchased from Biomiga 3 Use same volume of Wash Buffer 70 ethanol and Elution Buffer Biomiga EZgene Plasmid Midiprep 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 Genomic DNA Over time incubation Do not vortex or mix aggressively contamination after adding Buffer after adding buffer Bl Do not B1 incubate more than 5 minutes after adding Buffer B1 RNA contamination RNase A not added Add RNase A to Buffer A1 to Buffer Al Plasmid DNA floats out of wells while running in agarose gel DNA doesn t freeze or smell of ethanol Ethanol traces not completely removed
7. ions such as transfection of robust cells such as HEK293 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 Please contact our customer service for further information and reference Tablel for the commonly used plasmids Table 1 Commonly used plasmid and expected yield Plasmid Origin Copy Numbers Expected Yield ug per 50 mL pSC101 pSC101 5 5 pACYC PISA 10 12 5 10 pSuperCos pMB1 10 20 10 20 pBR322 pMB1 15 20 10 20 pGEM Muted pMB1 300 400 100 150 pBluescript 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 Page 2 Biomiga EZgene Plasmid Midiprep Kit 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
8. lumn from the tube and discard the flow through liquid Reinsert the column to the collection tube Repeat step 8 till all the lysate ethonal mix has been passed through the column Optional Add 3 0 mL Buffer KB into the spin column centrifuge at gt 2 500 x g for 1 minute Remove the spin column from the tube and discard the flow through Put the column back to the collection tube Note Buffer KB is recommended for endA strains such as HB101 JM101 TG1 or their derived strains It is not necessary for isolating DNA from endA strains such as Top 10 and DH5a Please reference Table 2 on page 3 Add 4 0 mL 70 ethanol into the column centrifuge at gt 2 500 x g for 1 min Remove the column from the tube and discard the flow through Reinsert the column into the collection tube Repeat step 10 Centrifuge the column with the lid open at gt 2 500 x g for 10 min to remove the ethanol residues Note Residual ethanol can be removed more efficiently with the column lid open High centrifuge speed is suggested to remove the ethanol It is critical to remove residual ethanol completely Carefully transfer the column into a sterile clean 15 mL tube and add 0 5 mL Elution Buffer or sterile ddH O to the center of the column and incubate for 1 min at room temperature Elute the DNA by centrifugation at gt 2 500 x g for 5 min For higher yield reload the eluate in the 15 mL tube to the column and incubate for 1 min Elute the
9. we recommend use product PD1712 Table2 endA strains of E Coli DH5a DHI DH21 JM106 JM109 SK2267 SRB XLO XLI1 TOP10 DHIOB JM103 JM107 SK1590 MM294 Stb 2 Ei ue XL10 BJ5182 DH20 JM105 JM108 SK1592 Select96 Stbl4 Goli o C600 JM110 RR1 ABLE C CJ236 KW251 P2392 BL21 DE3 ABLE BL21 DE3 HB101 TGl TBI DH12S LE392 PR700 K pLyss JM101 JM83 TKB1 HMS174 ES1301 M1061 Q358 BMH 71 18 All NM strains All Y strains Optimal Cell Mass OD 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 09 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 ODao A high ratio of biomass over lysis buffers result in low DNA yield and purity The midi column has an optimal biomass of 100 150 For example if the ODaoo is 3 0 the optimal culture volume should be 25 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 Al should be stored at 4 C once RNase A is added All other materials can be stored at room temperature
10. xing or pipetting Complete resuspension is critical for optimal yields 4 Add 2 5 mL Buffer B1 mix gently but thoroughly by inverting 5 times and incubate for 5 minutes to obtain a slightly clear lysate Note Do not incubate longer than 5 min Over incubating causes genomic DNA contamination and plasmid damage 5 Add 3 0 mL Buffer C1 mix immediately by inverting 5 times and sharp hand shaking for 3 times Note Adding ice cold C1 or incubating the lysate in ice will decrease the floating precipitates at step 6 Note It is critical to mix the solution well If the mixture still appears conglobated brownish or viscous more mix is required to completely neutralize the solution 6 Transfer the lysate to a high speed centrifuge tube and centrifuge at 14 000 x g for 10 minutes at room temperature Note If the rotor is cold incubate the lysate at room temperature for 10 minutes and then perform centrifugation as described 7 Carefully transfer the clear supernatant into a 15 mL tube avoid the floating precipitates Add 3 0 mL 100 ethanol Mix immediately by sharp hand Page 6 Biomiga EZgene Plasmid Midiprep Kit 10 11 12 13 shaking The mixture of ethanol lysate needs to be centrifuged through to the DNA column immediately Immediately transfer 6 0 mL the lysate ethonal mix into a DNA column with a 15 mL collection tube Centrifuge at gt 2 500 x g for 1 minute at room temperature Remove the co
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