Kit Manual
Contents
1. User Manual 1 1 1 Safety Information Buffer C1 and KB contains acetic acid wear gloves and protective eyewear when handling Biomiga EZgene Plasmid ezFilter Midiprep Kit II Page 5 EZgene Plasmid Midiprep II Spin Protocol 1 Inoculate 50 80 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 100 ml culture or cell mass greater than 250 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 5 mL Buffer Al Add RNase A into Buffer Al before use and completely resuspend bacterial pellet by vortexing or pipetting Complete resuspension is critical for optimal yields 4 Add 5 mL Buffer B1 mix gently but thoroughly by inverting 10 times and incubate for 5 minutes to obtain a slightly clear lysate Note Do not incubate longer than 5 minut2. by centrifugation at gt 2 500 x g for 5 minutes For higher yield reload the elute in the 15 mL tube to the columnand incubate for 1 min Elute the DNA again by centrifugation at gt 2 500 x g for 5 min 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 if the DNA is used for Biomiga EZgene Plasmid ezFilter Midiprep Kit II Page 7 endotoxin sensitive cell lines primary cultured cells or microinjection Note If ddH O is used for elution make sure that the pH is between 7 0 and 8 5 pH lower than 7 leads to lower elution efficiency 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 5 10 min Resuspend the DNA in Elution Buffer or sterile ddH O DNA concentration ug ml OD 44nm x 50 x dilution factor Page 8 Biomiga EZgene Plasmid ezFilter Midiprep Kit II Purification of Low Copy Number Plasmid and Cosmid The yield of low copy number plasmid is normally around 0 1 1 ug mL of overnight culture F
3. Add RNase A to buffer A1 to solution Al Plasmid DNA floats Ethanol traces not Make sure that no ethanol residual out of wells while completely removed remaining in the silicon membrane running in agarose from column before eluting the plasmid DNA gel DNA doesn t Re centrifuge or vacuum again if freeze or smell of necessary ethanol Page 10 Biomiga EZgene Plasmid ezFilter Midiprep Kit II
4. Contents Contents zoo AE E EE EEEE TOTE en 1 Introduction 3 2 io Feed v ree E E E E ede 2 Important Notes 5 cete ei proces a m ail e epON EDO P eee tbe 2 Storage and Stability sessie cece cece cece eee cece ence eens nene 3 Before Staring dodo ee reo eee uv oed tes Sto ERU 4 Kit Contents is cr Ix rede ie eene SE eee etae fee 5 Safety Information etis tp LEER REN SFR PII T tyre 5 EZgene Plasmid Midiprep II Spin Protocol 6 Purification of Low Copy Number Plasmid Cosmid 9 Trouble Shooting Guide sss 10 Biomiga EZgene Plasmid ezFilter Midiprep Kit II Page 1 Introduction Key to the kit is our proprietary DNA binding systems that allow the highly 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 no chaotropic salts are contained in the buffer of our patented plasmid purification kit The purified DNA is guanidine anion exchange resin residues free This kit is designed for fast and efficient purification of plasmid DNA from 50 to 100 mL of E coli culture The midi column has a plasmid DNA binding capacity of 500 ug The purified DNA is ready for high performance of downstream applications such as transfection of robust cells such as HEK293 restriction mapping library screening sequencing as well as gene the
5. LO TOP10 DHIOB JM103 JM107 SK1590 MM294 Stb12 ATA XL10 BJ5182 DH20 JM105 JM108 SK1592 Select96 Stbl4 Gold C600 JM110 RRI CJ236 KW251 P2392 BL21 DE3 HB101 TGI TBI ABLE DH12S LE392 PR700 BL21 DE3 K pLysS JM101 JM83 TKB1 HMS174 ES1301 M1061 Q358 BMH 71 18 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 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 ODs A high ratio of biomass over lysis buffers result in low DNA yield and purity The midi II column has an optimal biomass of 150 250 For example if the OD o9 is 3 0 the optimal culture volume should be 50 to 80 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 Biomiga EZgene Plasmid ezFilter Midiprep Kit II Pag
6. ds to be transferred to the DNA column immediately Immediately transfer the lysate ethonal mix into a DNA column with the collection tube Centrifuge at gt 2 500 x g for 1 minutes at room temperature Remove the column 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 4 0 mL Buffer KB into the spin column centrifuge at 2 500 x g for 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 5 mL 70 ethanol into the column centrifuge at gt 2 500 x g for 1 minutes 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 2 500 x g for 10 minutes to remove the ethanol residues Note Residual ethanol can be removed more efficiently with the column lid open It is critical to remove residual ethanol completely Carefully transfer the spin column to a sterile clean tube and add 0 5 1 mL ddH O or Elution Buffer to the center of the column and incubate for 1 minute at room temperature Elute the DNA
7. e 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 A1 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 Incubate Buffer C1 at 4 C before experiment It will decrease the floating precipitate Keep the cap tightly closed for Buffer B1 after use Make sure the availability of centrifuge 13 000 rpm 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 50 mL conical tubes Isopropanol if precipitate the plasmid DNA Page 4 Biomiga EZgene Plasmid ezFilter Midiprep Kit II Kit Contents Catalog PD1414 00 PD1414 01 PD1414 02 Preps 2 10 25 ezBind Columns 2 10 25 jor ge 2 10 25 Buffer Al 11 mL 55 mL 135 mL Buffer B1 11 mL 55 mL 135 mL Buffer C1 14 mL 70 mL 170 mL Buffer KB 9 mL 50mL 110 mL RNase A 20 mg mL E uL ds uL ae uL Elution Buffer 4mL 20 mL 60 mL
8. es Over incubating causes genomic DNA contamination and plasmid damage 5 Add 6 mL Buffer C1 mix immediately by inverting 5 times and vortex for 10 seconds 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 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 50 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 i Hold the filter syringe barrel over the 50 mL tube and gently insert t plunger to expel the cleared lysate to the tube stop when feel major resistance Page 6 Biomiga EZgene Plasmid ezFilter Midiprep Kit II 10 11 12 13 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 Add 6 mL 100 ethanol Mix immediately by sharp shaking The mixture of ethanol lysate nee
9. or isolating low copy number or medium copy number plasmid DNA use the following guideline 1 Culture volume Use 2 x volume of the high copy number culture Use up to 160 mL for the midiprep II 2 Use2 x volume 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 Midiprep Kit II Page 9 Trouble Shooting Guide Problem Possible Reason Suggested Improvement 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 2M 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 the plasmid volume of buffer Al B1 C1 and 100 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 Bl incubate more than 5 minutes after adding solution B1 RNA contamination RNase A not added
10. rapy 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 Table 1 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 P15A 10 12 5 10 pSuperCos pMB1 10 20 10 20 pBR322 pMB1 15 20 10 20 pGEM Muted pMB1 300 400 100 150 pBluescript CoIEI 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 TGI1 and their derivatives normally have low plasmid yield due to either endogenous endonucleases or high Page 2 Biomiga EZgene Plasmid ezFilter Midiprep Kit II 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 DH5a DHI DH21 JM106 JM109 SK2267 SRB X
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