Kit Manual
Contents
1. 1 5 mL DNA 50 100 puL gt 50 uL ddH2O pH 7 0 8 5 Elution Buffer 2 13 000 rpm 1 DNA 13 000 rpm 1 YE DNA HEK293 PD1212 o Biomiga EZgene Plasmid Miniprep Kit Page 11 Trouble Shooting Guide Problems Possible Reasons Suggested Improvements Low Yield Poor Cell lysis e Resuspend pellet thoroughly by votexing and pipetting prior to adding Buffer B1 e Make fresh Buffer B1 if the cap had not been close2. ODeoo re 1 4 mL LB a gallate WEKE cs TB 2xYT ODsoo 3 0 2 0 3 0 cs o 37 C 5 LB Luria Bertani 12 16 2 5 250 uL Buffer A1 RNase A W 250 uL Buffer B1 5 10 Page 10 Biomiga EZgene Plasmid Miniprep Kit 10 5 BNA
3. Buffer B1 350 uL Buffer N1 ERIRE 13 000 rpm 10 DNA 13 000 rpm 1 DNA 500 uL Buffer KB 13 000 rpm 1 YE end4 HB101 JM101 TG1 endA Top 10 DH5a 3 2 650 pL DNA Wash Buffer 13 000 rpm 1 8 13 000 rpm 2
4. SK1592 Select96 Stbl4 ae C600 JM110 RRI ABLE C CJ236 KW251 P2392 BL21 DE3 HB101 TGI TB1 ABLE K DHI2S LE392 PR700 as JM101 JM83 TKB1 HMS174 ES1301 M1061 Q358 BMH 71 18 All NM strains All Y strains Optimal Cell Mass OD6o_ 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 mini column has an optimal biomass of 10 15 For example if the ODao is 3 0 the optimal culture volume should be 1 5 mL For over amount of cell numbers either reduce the biomass or scale up the volumes of Buffer Al B1 and N1 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 Plasmid Miniprep Kit Page 3 Bef
5. User Manual 1 1 1 1 Add 8 mL PD1211 00 or 60 mL PD1211 01 or 96 mL PD1211 02 or 60 mL PD1211 03 96 100 ethanol to each DNA Wash Buffer bottle before use Safety Information e Buffer N1 contains acidic acid wear gloves and protective eyewear when 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 Page 5 EZgene Plasmid Miniprep Spin Protocol 1 Inoculate 1 4 mL LB containing appropriate antibiotic with a fresh colony from a freshly streaked selective plate 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 ODs Buffers need to be scaled up proportionally if over amount of cultures are being processed 2 Harvest the 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 Resid
6. Contents LEO NES RT ee A AEE Introduction sane ed Dn EE TA E E E EE E Important Notes 2 228i daca sealed ali a oe Me Mel Mad eueaied aki ad Storage and Stability ccc cece cece eee e tees ee enes Before Startine sae np yoade sang seeae bese a E a Ea Kit Contents lt u iscsi occupa A de ee Safety Information s 3 ee en a i a poda dai EZgene Plasmid Miniprep Spin Protocol pp EZgene Plasmid Miniprep Spin Vacuum Protocol 6 Purification of Low Copy Number Plasmid and Cosmid DENAN o AL Trouble Shooting Gulde 10 12 Biomiga EZgene Plasmid Miniprep Kit Page 1 Introduction Key to the kit is our proprietary DNA binding systems that allow the high efficient reversible binding of DNA to the mini column while proteins and other impurities are removed by wash buffer Nucleic acids are then eluted with sterile water or elution buffer This kit is designed for fast and efficient purification of plasmid DNA from to 4 mL of E coli culture The mini column has a plasmid DNA binding capacity of 50 ug The yield from 1 mL culture is typically around 8 to 12 ug Plasmid Miniprep Kit I PD1213 with the plasmid DNA binding capacity of 80 ug is recommended if higher yield gt 50 ug is desired The purified DNA is ready for downstream applications such as cloning subcloning RFLP sequencing and transfection of robust cells such as HEK293 cells Important Notes Plasmid Copy N
7. Page 9 RNase A 4 C RNase A Buffer Al I C PD1211 I 4 C DNA Wash Buffer Buffer Al RNase AH F 8 mL PD1211 00 60 mL PD1211 01 96 mL PD1211 02 60 mL PD1211 03 96 100 DNA Wash Buffer Buffer B1 Buffer B1 AYR ye a HIER o 22 25 C ll A1 B1 N1 Wash Buffer Elution Buffer Pete bal 70 C 50 C 2 2 Buffer AR UI BRE RR 1 14 16 10 000 x
8. d 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 and the plasmid volume of Buffer Al B1 N1 as instructed on page 9 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 A1 Plasmid DNA floats Ethanol traces were Make sure that no ethanol residue out of wells while not completely remains in the silicon membrane running in agarose removed from before elute the plasmid DNA Re gel DNA _ doesn t column centrifuge or vacuum again if freeze or smell of ethanol necessary Page 12 Biomiga EZgene Plasmid Miniprep Kit
9. ications such ascloning 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 PD1212 if the DNA is used for endotoxin sensitive cell lines primary cultured cells or microinjection The DNA concentration can be calculated as follows Concentration ug mL OD260nm X 50 x dilution factor Biomiga EZgene Plasmid Miniprep Kit Page 7 EZgene Plasmid Miniprep Spin Vacuum Protocol 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 isolating plasmid from 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 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 to improve the recovery Transfer the column with the lid open to a 1 5 mL collection tube and centrifuge at 13 000 rpm for 2 minutes Carefully transfer the s
10. nd put the column back to the collection tube Optional Add 500 uL Buffer KB into the spin column centrifuge at 13 000 rpm 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 DHSa 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 minute at room temperature Remove the spin column from the tube and discard the flow through Repeat step 9 to improve the recovery Reinsert the spin column with the lid open into the collection tube and 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 sterile 1 5 mL tube and add 50 100 uL gt 50 uL Sterile ddH 0 or Elution Buffer into the center of the column and let it stand for 2 minutes Elute the DNA by centrifugation at 13 000 rpm for 1 minute Reload the eluate into the column and elute again Note 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 ddH2O Note The DNA is ready for downstream appl
11. ore Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps and pay Special attention to the followings Important e RNase A 20 mg mL 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 Add8 mL PD1211 00 or 60 mL PD1211 01 or 96 mL PD1211 02 or 60 mL PD1211 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 user e High speed microcentrifuge or Vacuum manifold e 96 100 ethanol e 1 5 mL microcentrifuge tubes Page 4 Biomiga EZgene Plasmid Miniprep Kit Kit Contents Catalog PD1211 00 PD1211 01 PD1211 02 PD1211 03 Preps 4 50 250 100 ezBind Columns 4 50 250 100 Buffer Al 1 2 mL 15 mL 70 mL 28 mL Buffer B1 1 2 mL 15 mL 70 mL 28 mL Buffer N1 1 6 mL 20 mL 100 mL 40 mL Buffer KB 3 mL 30 mL 135 mL 55 mL DNA Wash Buffer 2 mL 15 mL 3 x 24 mL 2x15 mL Elution Buffer 600 uL 10 mL 30 mL 30 mL RNase AQOme mL Gout 5 soul don
12. pin column into a clean 1 5 mL tube and add 50 100 uL gt 50 uL Sterile ddH 0 or Elution Buffer into the column and let it stand for 2 minutes Elute the DNA by centrifugation at 13 000 rpm for 1 minute Reload the eluate into the column and elute again 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 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 Culture volume Use 2 x volumes of the high copy number culture 2 Use 2 x volumes of the Buffer A1 Buffer B1 and Buffer N1 Additional buffers can be purchased from Biomiga 3 Use same volume of Wash Buffer DNA Wash Buffer and Elution Buffer Purification of plasmid gt 12 kb For isolating plasmid DNA gt 12 kb use the following guideline 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 Use same 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 minutes after adding Elution Buffer Biomiga EZgene Plasmid Miniprep Kit
13. ue medium will cause e Poor cell lysis and thus lower DNA yield e Loose pellet after centrifugation in step 6 3 Add 250 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 4 Add 250 uL Buffer B1 mix gently by inverting the tube 10 times do not vortex and incubate at room temperature for 5 minutes Note Do not incubate for more than 5 minutes Note Buffer B1 precipitates cloudy look below room temperature Warm up Buffer B1 at 50 C to dissolve precipitation before use 5 Add 350 uL Buffer N1 mix completely by inverting shaking the vial for 5 times and sharp hand shaking for 2 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 6 Centrifuge the lysate at 13 000 rpm for 10 minutes at room temperature Page 6 Biomiga EZgene Plasmid Miniprep Kit 10 11 12 Note If the lysate doesn t appear clean reverse the tube angle centrifuge for 5 more minutes and then transfer the clear lysate to DNA column Carefully transfer the clear lysate into a DNA column with a collection tube avoid the precipitations spin at 13 000 rpm for minute discard the flow through a
14. umbers 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 plasmids and expected yield Plasmid Origin Copy Numbers Expected Yield ug per 1 mL pSC101 pSC101 5 0 1 0 2 pACYC P15A 10 12 0 4 0 6 pSuperCos pMB1 10 20 0 4 1 pBR322 pMB1 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 Please reference Table 2 for the endA information Page 2 Biomiga EZgene Plasmid Miniprep Kit Table 2 endA strains of E Coli DHSa DHI DH21 JM106 JM109 SK2267 SRB XLO TOP10 DH10B JM103 JM107 SK1590 MM294 Stbl2 a BJ5182 DH20 JM105 JM108
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