Kit Manual
Contents
1. n BJ5182 DH20 JM105 JM108 SK1592 Select96 Stbl4 Fare EndA Strains of E Coli C600 JM110 RRI ae CJ236 KW251 P2392 BL21 DE3 HB101 TGI TB1 ABLE DH12S LE392 PR700 BEDE K pLysS JM101 JM83 TKB1 HMS174 ES1301 M1061 Q358 BMH 71 18 All NM strains All Y strains Optimal Cell Mass ODs 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 o0 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 ODo00 A high ratio of biomass over lysis buffers result in low DNA yield and purity The column has an optimal biomass of 150 250 For example if the ODeoo is 3 0 the optimal culture volume should be 100 to 200 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 22 25 C The Guaranteed shelf life is 12 months from the date of purchase Biomiga EZgene Express Plasmid Maxiprep Kit Page 3 Before Starting Prepare all c2. 15 mL 54 mL 2x54mL Nee a i Conn neon Elution Buffer 6 mL 30 mL 75mL User Manual 1 1 1 Add 60 mL PD1568 00 or 216 mL PD1568 01 or 216 mL PD1568 02 96 100 ethanol to each DNA Wash Buffer bottle before use Safety Information e Buffer C1 contains acetic acid wear gloves and protective eyewear when handling e Buffer C1 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 Express Plasmid Maxiprep Kit Page 5 EZgene Express Plasmid Maxiprep Protocol 1 Inoculate 100 200 mL LB containing appropriate antibiotic with 100 ul fresh starter culture Grow 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 Warning Do not use more than 200 ml culture Need to scale up buffers if processing more than 100 mL culture Harvest the bacterial by centrifugation at 5 000 g for 10 min at room temperature Pour off the supernatant and blot the inverted tube on paper towels to remove residual medium Note Complete removal of residue medium is critical for bacteria lysis in the next step Add 10 mL Buffer Al and completely resuspend bacterial pellet by vortexing or pipett
3. elution normally yields about 60 70 of the DNA and the second elution yields another 20 30 of the DNA Note The DNA is ready for downstream applications such as cloning or transfection of HEK293 cells Note It s highly recommended to remove the endotoxin if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection Please use Biomiga s Endofree Plasmid Midiprep Kit PD1520 and PD1522 for transfecting endotoxin sensitive cell lines primary cultured cells or microinjection Biomiga EZgene Express Plasmid Maxiprep Kit Page 7 Express Plasmid Maxiprep Flow Chart Lysis and neutralization Clear Lysate through a Syringe filter e a ay Bind DNA to the DNA column E F m E Wash the column with DNA Wash Buffer by a plunger Detach the column and insert to a 1 5 mL tube Spin the column to dry and elute the DNA Page 8 Biomiga EZgene Express Plasmid Maxiprep Kit 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 fresh at 20 C if the culture is not purified the same
4. Contents Contents aoe A ner aoa eee roe eos eon eset Ee E 1 Introductions eree een e eda sea a E stan E 2 Important iNote s n E ner een anaes 2 Storagetand Stability ee a e ee ee 3 BetorerStartin Caan cee en 4 GTN Croli lilo eran sananana E E E N O E stg atads 5 Safety ntornmati Orna a aE E E 5 EZgene Plasmid Maxiprep Spin Protocol cccccceceeeee eee ees 6 Express Plasmid Maxiprep Flow Chatt eccceceececcceteeeeeeneenees 8 Mrouble Shootin CUE E E 9 Biomiga EZgene Express Plasmid Maxiprep Kit 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 100 to 200 mL of E coli culture The midi column has a plasmid DNA binding capacity of 1000 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 therapy and genetic vaccinations Important Notes Plasmid Copy Numbers The yield of p
5. 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 Add RNase A to buffer Al to solution A1 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 Biomiga EZgene Express Plasmid Maxiprep Kit Page 9
6. ing Ensure that RNase A has been added into Buffer Al before use Note Complete resuspension is critical for optimal yield Add 10 mL Buffer B1 mix thoroughly by inverting 10 times with gentle shaking Incubate for 5 10 min to obtain a slightly clear lysate Complete lysis is critical for DNA yield The mixture of completely lysed bacteria looks transparent Attention Buffer Bl forms precipitation below room temperature if solution becomes cloudy warm up at 37 C to dissolve before use Add 12 Buffer C1 mix completely by inverting the tube 10 times and shaking for 5 times 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 Two options for clearing the lysates High speed centrifuge Transfer the lysate to a high speed centrifuge tube and centrifuge at 13 000 rpm 14 000 18 000 g for 15 min at room temperature Transfer the cleared lysate to a 50 ml conical tube ezFilter syringe Pour the lysate into the barrel of the filter syringe and set the syringe ina 50 mL conical tube Incubate at room temperature for 10 min The white precipitates should float to the top Gently insert Page 6 Biomiga EZgene Express Plasmid Maxiprep Kit 10 11 12 13 14 the plunger to expel the cleared lysate to the tube stop when feel strong resistance some of the lysate may remain in the flocculent precipitate Note T
7. lasmid 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 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 Page 2 Biomiga EZgene Express Plasmid Maxiprep Kit 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 DH5a DHI DH21 JM106 JM109 SK2267 SRB KO TOP10 DH10B JM103 JM107 SK1590 MM294 Stbl2
8. o avoid clog of the syringe Use less than 200 mL of overnight culture and mix the lysate well after adding Buffer C1 Alternatively transfer the lysate to another syringe filter Additional syringe filter can be purchased from Biomiga separately Add 12 mL absolute ethanol 96 100 to the cleared lysate Mix well by sharp shaking for 5 times Add the lysate ethanol mixture into a DNA column set in a 50 mL conical tube Use the plunger to expel the lysate through the column Gently pull the plunger out add 10 mL DNA Wash Buffer expel the Buffer out with the plunger Expel the liquid completely by push and pull of the plunger several times Repeat step 9 Use the plastic wrench Provided to detach the end component from the midiprep column and insert it into a 1 5 ml eppendorf tube Spin the column at 13 000 15 000 rpm Max speed for 1 min Decant the flow through and put the column back to the tube Spin the column at max speed for 2 min Add 500 pL Elution Buffer Prewarm the Elution Buffer 60 C increases the yield to the center of the column and incubate for 1 min at room temperature Elute the DNA by centrifugation at 12 000 rpm for 1 min Then add 300 uL Elution Buffer to the center of the column and incubate for 1 min at room temperature Elute the DNA by centrifugation at 12 000 rpm for 1 min Repeat step 12 to improve the recovery Optional Add the eluted DNA back to the column for another elution The first
9. omponents 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 Buffer C1 may form precipitates below 10 C warm up at 37 C to dissolve the precipitates before use 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 by centrifugation Carry out all centrifugations at room temperature For certain reasons Buffer KB are not involved in this kit from now on Materials supplied by users 100 ethanol High speed centrifuge 50 mL high speed centrifuge tubes 50 mL conical tubes Isopropanol if precipitate the plasmid DNA Page 4 Biomiga EZgene Express Plasmid Maxiprep Kit Kit Contents Catalog PD1568 00 PD1568 01 PD1568 02 Preps 2 10 DS ezBind Columns 2 10 25 60m yeas 2 10 25 1 5ml Microfuge tube 4 20 50 Plastic wrench 1 1 1 Buffer Al 22 mL 110 mL 270 mL Buffer B1 22 mL 110 mL 270 mL Buffer C1 27 mL 135 mL 2x170mL DNA Washing Buffer
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