Kit Manual
Contents
1. 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 3 to 5 times Please reference Table 1 for the commonly used plasmids Table 1 commonly used plasmid and expected yield Plasmid Origin Copy Numbers Expected Yield ug per 1 mL pSC101 pSC101 3 0 1 0 2 pACYC PISA 10 12 0 4 0 6 pSuperCos pMB1 10 20 0 4 1 pBR322 pMBI 15 20 0 6 1 pGEM Muted pMB1 300 400 6 7 pBluescript ColE1 300 500 6 8 pUC Muted pMB1 500 700 8 12 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 Page 2 Biomiga EZgene Plasmid Miniprep Kit II Table2 endA strains of E Coli DH5a DHI DH2 JMI106 JM109 SK2267 SRB XLO TOPI0 DHIOB JMIO3 M107 SK1590 MM294 swm XE BI5182 DH20 JMIOS IMIOS SKIS92 Select96 Suam SEP C600 JM110 RRI ABLE C CJ232. DNA gt 12 kb use the following guideline 1 Culture volume Use 2 x volumes of the culture 2 Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer N1 Additional buffers can be purchased from Biomiga 3 Usesame volume of Wash Buffer DNA Wash Buffer and Elution Buffer 4 Pre warm the Elution Buffer at 65 70 C and let the column stand for 5 mins after adding Elution Buffer Biomiga EZgene Plasmid Miniprep Kit II Page 9 Trouble Shooting Guide Problems D Suggested Improvements Low Yield Bacterial Grow bacterial 12 16 hours Spin down culture cultures and store the pellet at 20 C if the overgrown or culture is not purified the same day Do not not fresh store culture at 4 C over night Low Yield Low copy Increase culture volume and scale up the number volume of buffers according to the plasmid instruction on page 9 No DNA Plasmid lost Prepare fresh culture in Host E coli Genomic DNA Over time Do not vortex or mix aggressively after contamination incubation adding Buffer B1 Do not incubate more than after adding 5 minutes after adding Buffer B1 buffer B1 RNA RNase A not Add RNase A to Buffer Al contamination added to solution Al Plasmid DNA Trace EtOH Make sure that no ethanol residues remain in floats out of not the silicon membrane before elute the wells while completely plasmid DNA Re centrifuge or vacuum running in removed from again if ne
3. DNA column with a collection tube avoid the precipitations spin at 13 000 rpm for 1 minute discard the flow through and put the column back to the collection tube Carefully transfer the remaining clear lysate to the column and centrifuge at 13 000 rpm for 1 minute at room temperature and discard the flow through in the collection tube Put the column back to the collection tube Optional Add 500 uL Buffer KB into the spin column centrifuge at 13 000 rpm 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 650 uL DNA Wash Buffer Add ethanol to DNA wash buffer before use into the spin column centrifuge at 13 000 rpm for 1 minute at room temperature Remove the spin column from the tube and discard the flow through Repeat step 9 to improve the recovery 10 Reinsert the spin column with the lid open into the collection tube and 11 centrifuge for 2 minutes at 13 000 rpm 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 into a clean 1 5 mL tube and add 100 150 uL ddH 0 or Elution Buffer into the center of the co
4. 6 KW251 P2392 BL21 DE3 c HB101 TG1 TB1 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 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 ODs 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 OD 00 A high ratio of biomass over lysis buffers result in low DNA yield and purity The mini column has an optimal biomass of 30 45 For example if the OD600 is 3 0 the optimal culture volume should be 10 15 mL For over amount of cell numbers either reduce the biomass or scale up the volumes of Buffer Al B1 and NI Culture Volume Use a flask or tube 4 times bigger in volumn thanthe 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 Plasmid Miniprep Kit II Page 3 Before Starting Prepare all components and get all necessary materials ready by examining this instruct
5. Table of Contents Contents co ie tb ero EE REIR Rr EE PURSE ERE Me E sets 1 Introduction e ecc d rt HERR sevens a beteenz ia a gt 2 Important NNOteS nete per cote ete tecto imde Lou E eub 2 Storage and Stability neon e Rer ERO 4 Before Starting epe te ui ebrei Reed 4 Kit Contents c ecc tete toe eee e RR Eee 5 Safety Information s is cases onbe tn dtu peste gehen eR AX RO E deos 5 EZgene Plasmid Miniprep II Spin Protocol eese 6 EZgene Plasmid Miniprep II Spin Vacuum Protocol 8 Purification of Low Copy Number Plasmid Cosmid 9 Trouble Shooting Guide esee 10 Biomiga EZgene Plasmid Miniprep Kit II 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 contaminates are removed under certain optimal conditions Nucleic acids are easily eluted with sterile water or Elution Buffer This kit is designed for fast and efficient purification of plasmid DNA from 1 to 15 mL of E coli culture With the binding capacity of 80 ug the yield obtained by Miniprep Kit II PD1213 is higher than Miniprep Kit I PD1211 The yield from 1 mL culture is typically around 8 to 12 ug The purified DNA is ready for downstream applications such as cloning subcloning RFLP sequencing and transfection of HEK293 cells Important Notes Plasmid Copy Numbers
6. at 6 000 rpm in a 15 mL conical tube for 10 minutes if high speed centrifuge tubes are not available Alternatively the cultures can also be spin down in multiple 2 0 mL tubes Add 450 uL Buffer A1 Add RNase A to Buffer A1 before use and completely resuspend bacterial pellet by vortexing or pipetting Note Complete resuspension is critical for bacterial lysis and lysate neutralization Add 450 uL Buffer B1 mix gently by inverting 10 times do not vortex and incubate at room temperature for 5 minutes If necessary continue inverting the tube until the solution becomes slightly clear Note Do not incubate for more than 5 minutes Note Buffer B1 precipitates cloudy look below room temperature Warm up Buffer B1 at 37 C to dissolve precipitation before use Add 550 uL Buffer N1 mix completely by inverting shaking the vial for 5 times and sharp hand shaking for 3 times Note Incubating the lysate in ice for 1 min will improve the yield Note It is critical to mix the solution well If the mixture still appears conglobated brownish or viscous more mixing is required to completely neutralize the solution Centrifuge the lysate at 13 000 rpm for 10 minutes at room temperature Note If the lysate doesn t appear clean reverse the tube angle centrifuge for 5 more Page 6 Biomiga EZgene Plasmid Miniprep Kit II minutes and then transfer the clear lysate to DNA column Carefully transfer up to 700 uL clear lysate into a
7. cessary agarose gel column DNA doesn t freeze or smell of ethanol Page 10 Biomiga EZgene Plasmid Miniprep Kit II
8. ion booklet and become familiar with each steps Important e RNase A It is stable for more than half a year when stored at room temperature Spin down RNase A vial briefly Add the RNase A solution to buffer Al and mix well before use Store at 4 C e Add 8 mL PD1213 00 or 60 mL PD1213 01 or 96 mL PD1213 02 or 60 mL PD1213 03 96 100 ethanol to each DNA Wash Buffer bottle before use 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 Keep the cap tightly closed for Buffer B1 after use e Ensure the availability of centrifuge capable of 13 000 rpm e Carry out all centrifugations at room temperature Materials supplied by users e 96 100 ethanol e 1 5 mL 2 0 mL microcentrifuge tubes e 5mL conical tubes e High speed microcentrifuge or Vacuum manifold Page 4 Biomiga EZgene Plasmid Miniprep Kit II Kit Contents Catalog PD1213 00 PD1213 01 PD1213 02 PD1213 03 Preps 4 50 250 100 ezBind Columns 4 50 250 100 Buffer Al 2 5 mL 25 mL 125 mL 50 mL Buffer B1 2 5 mL 25 mL 125 mL 50 mL Buffer NI 3 mL 30 mL 135 mL 60 mL Buffer KB 3 mL 30 mL 135 mL 60 mL DNA Wash Buffer 2 mL 15 mL 3x 24mL 2x15 mL Elution Buffer 1 mL 15 mL 60 mL 30 mL ta 23 ul ub i oi User Manual 1 1 1 1 Add 8 mL PD1213 00 or 60 mL PD1213 01 or 96 mL PD1213 02 or 60 mL PD1213 03 96 100 ethan
9. lumn and let it stand for 2 minute Elute the DNA by centrifugation at 13 000 rpm for 1 minute Reload the eluate into the column and centrifuge again to improve the recovery Note The pH of Elution Buffer or ddH5O will affect the plasmid DNA elution If ddH O 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 cells such as HEK293 cells Note It s highly recommended to remove the endotoxin PD1214 if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection 12 The DNA concentration can be calculated as Concentration ug mL OD nm x 50 x dilution factor Biomiga EZgene Plasmid Miniprep Kit II Page 7 EZgene Plasmid Miniprep II Spin Vacuum Protocol 1 Set up the vacuum manifold according to manufacture s instruction and connect the column to the manifold Carry out step 1 6 on Page 6 in previous protocol Carefully transfer the clear lysate to the DNA column and turn on the vacuum to allow the lysate pass through the column Optional Add 500 uL Buffer KB into the spin column and allow the lysate pass through the column by vacuum Note Buffer KB is recommended for endA strains such as HB101 JM101 TG1 or their derived strains It is not necessa
10. ol to each DNA Wash Buffer bottle before use Safety Information e Buffer N1 contains acidic acid wear gloves and protective eyewear while handling e Buffer N1 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 Miniprep Kit II Page 5 EZgene Plasmid Miniprep II Spin Protocol For 1 4 mL culture reduce the volume of Buffer A1 B1 N1 to 250 uL 250 uL and 350 uL respectively And use the same volume of DNA Wash Buffer and Elution Buffer 1 Inoculate 5 12 mL LB containing appropriate antibiotic with a fresh colony Incubate at 37 C for 14 16 hours with vigorous shaking Note Prolonged incubation gt 16 hours is not recommended since the E coli starts to lyse and the plasmid yields may be reduced Note Do not grow the culture directly from the glycerol stock Note This protocol is optimized for E coli strain cultured in LB medium When using TB or 2xYT medium special care needs to be taken to ensure the cell density doesn t exceed 3 0 OD600 Buffers need to be scaled up proportionally if over amount of cultures are being processed Harvest bacterial culture by centrifugation for 1 min at 10 000 rpm Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium Remove the residue medium completely Note The culture can be centrifuged
11. ry for isolating DNA from endA strains such as Top 10 and DH5a Please reference Table 2 on page 3 Add 650 uL of DNA Wash Buffer to the column and allow the vacuum to draw the liquid through the manifold Turn off the vacuum Repeat step 5 Transfer the column with the lid open to a 2 mL collection tube and centrifuge at 13 000 rpm for 2 minutes Carefully transfer the spin column into a clean 1 5 mL tube and add 100 150 uL ddH 0 or Elution Buffer into the column and let it stand for 1 minute Elute the DNA by centrifugation at 13 000 rpm for 1 minute Reload the eluate into the column and centrifuge again to improve the recovery Note The DNA is ready for downstream applications such as cloning RFLP library screening in vitro translation sequencing and transfection of robust cells such as HEK293 cells Page 8 Biomiga EZgene Plasmid Miniprep Kit II 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 the high copy number culture Use 25 mL for the miniprep II 2 Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer N1 Additional buffers can be purchased from Biomiga 3 Usesame volume of Wash Buffer DNA Wash Buffer and Elution Buffer Purification of plasmid gt 12 kb For isolating plasmid
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