Kit Manual
Contents
1. of wells while completely removed remaining in the silicon membrane before elute the plasmid DNA Re centrifuge or vacuum again if necessary No phase partitioning after centrigugation Temperature is lower than 23 C Make sure the temperature is greater than 23 C for centrifugation or incubate the sample at 60 C for 5 min and then perform centrifugation Page 10Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II2. 1 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 Table2 endA strains of E Coli DH5a DHI DH21 JM106 JM109 SK2267 SRB XLO TOP10 DHIOB JM103 JMIO7 SK1590 MM294 Stb 2 XLI Blue XL10 BJ5182 DH20 JM105 JM108 SK1592 Select96 Stbl4 Gold C600 JM110 RRI ABLE C CJ236 KW251 P2392 BL21 DE3 HB101 TGI TBI ABLE K DES LE392 PR700 ur aa JM101 JM83 TKBl1 HMS174_ ES1301 M1061 Q358 BMH 71 18 All NM strains All Y strains Optimal Cell Mass OD o9 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 medium such as TB or 2xYT are used make sure the cell density doesn t exceed 3 0 ODgo9 A high ratio of biomass over lysis buffers result in low DNA yield and purity The mini column II has an optimal biomass of 10 36 For example if the OD6oo is 3 0 the optimal culture volume should be 3 12 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
3. Table of Contents Introduction toutes aeter pro ETE arbe tef Tete b igh 2 Important Notes een etes Mrs rte Ex erae esL ve ou Re ds 2 Storage and Stability ccc cece cece eee eee a eee eene 3 Before Starting core ege Fe tace ste dude e Rie ee stie d ve panied ds 4 Kit Contents ceder eile eet mete etd eet ee de ded boe 5 Safety Information sss enmt mener 5 EZgene EndoFree Plasmid ezFlow Miniprep II Spin Protocol 6 EZgene EndoFree Plasmid ezFlow Miniprep II Spin Vacuum gisrevo MERE Purification of Low Copy Number Plasmid and Cosmid 9 Trouble Shooting Guide sese eee 12 Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II 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 Plasmid isolated with traditional protocol normally contains high level of endotoxins lipopolysaccharides or LPS For transfection of endotoxin sensitive cell lines or microinjection the endotoxins should be removed before the applications The EZgene endofree system uses a specially formulated buffer that extracts the endotoxin from the plasmid DNA Two rounds of extraction will reduce the endotoxin level to 0 1 EU Endotoxin per ug of plasmid DNA The endofre
4. a 1 5 mL tube and add 50 100 uL of Endofree Elution Buffer Incubate for 1 minute at room temperature and centrifuge at 13 000 rpm for 1 minute to elute DNA Reload the eluate into the column use the same 1 5 mL tube and incubate for minute centrifuge at 13 000 rpm for 1 minute to elute DNA Note The eluted DNA is ready for transfection of endotoxin sensitive cell lines primary cultured cells or microinjection The DNA concentration can be calculated as follows DNA concentration ug mL OD260nm x 50 x dilution factor Note Two elutions give rise to maximum DNA yield Use less Endofree Elution Buffer if high concentration is desired Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II Page 7 EZgene Plasmid ezFlow Miniprep 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 7 in previous protocol on page 6 and 7 Carefully transfer the solution from step 7 in the previous protocol to a DNA column and turn on the vacuum to allow the lysate pass through the column Repeat until the remaining solution pass through the column Add 500 uL Buffer KB to the column and allow the buffer pass the column by vacuum Note This step is important to remove residual protein contaminations especially for endA strains and be highly recommended for high qual i ty plasmid DNA Add 650 uL of DNA Wash Buffer to the column and allo
5. als supplied by users e 96 100 ethanol e 1 5 mL and 2 0 mL microcentrifuge tubes e High speed microcentrifuge or Vacuum manifold Page 4 Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II Kit Contents Catalog PD1222 00 PD1222 01 PD1222 02 Preps 4 50 250 ezBind Columns 4 50 250 Buffer Al 2 5 mL 25 mL 125 mL Buffer B1 2 5 mL 25 mL 125 mL Buffer N3 400 uL 5 mL 25 mL Buffer KB 2 5 mL 30 mL 135 mL Buffer RET 4 mL 50 mL 250 mL DNA Wash Buffer 2 mL 15 mL 3 x 24 mL Endofree Elution Buffer 1 mL 10 mL 30 mL RNase A 20 mg mL a p dos ms ur T User Manual 1 1 1 Add 8 mL PD1222 00 or 60 mL PD1222 01 or 96 mL PD1222 02 96 100 ethanol to each DNA Wash Buffer bottle before use Safety Information e Buffer N3 contain acetic acid wear gloves and protective eyewear when handling e Buffer N3 KB and RET 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 EndoFree ezFlow Plasmid Miniprep Kit II Page 5 EZgene Plasmid ezFlow Miniprep Spin Protocol 1 Inoculate 3 12 mL LB containing appropriate antibiotic with a single colony from a freshly streaked selective plate Grow at 37 C for 14 16 hours with vigorous shaking Note Do not use a streaked plate that has been stored at 4 C Note Do not inoculate culture directly with glyce
6. e plasmid miniprep kit provides an efficient endotoxin removal step into the traditional purification procedure to produce transfection grade plasmid DNA This kit is designed for fast and efficient purification of plasmid DNA from 3 to 12 mL of E coli culture The mini column II has a DNA binding capacity of 80 ug The purified endofree DNA is ready for downstream applications such as transfection of endotoxin sensitive cell lines primary cultured cells or microinjection 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 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 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 Page 2 Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II 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 HB10
7. ly transfer about 750 uL clear lysate no more than 800 uL to a clean 2 0 mL tube and add 1 volume of Buffer RET For example 800 Page 6 Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II 10 11 12 13 uL of Buffer RET to 800 pL of clear lysate and 400 uL of 100 ethanol Mix well by sharp hand shaking for 3 times Transfer 700 uL of the lysate ethanol mixture to a DNA spin column and centrifuge at 13 000 rpm for 20s Discard the flow through liquid and transfer the remaining lysate ethanol mixture to the column Centrifuge at 13 000 rpm for 30s and discard the flow through put the column back to the collection tube 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 This step is important to remove residual protein contaminations especially for endA strains and be highly recommended for high qual i ty plasmid DNA Add 650 uL DNA Wash Buffer and centrifuge at 13 000 rpm for 20s Discard the flow through liquid and insert the column with the lid open back to the collection tube Repeat step 10 Centrifuge the column with the lid open at 13 000 rpm for 5 10 minutes to remove the residual ethanol Note Residual ethanol can be removed more efficiently with the column lid open It is critical to remove residual ethanol completely Transfer the column to
8. 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 EndoFree ezFlow Plasmid Miniprep Kit II Page 3 Before Starting Two endotoxin removal procedures are provided Protocol A removes endotoxin during the purification of plasmid DNA and Protocol B removes endotoxin after the purification of plasmid DNA 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 It is stable for half a year under room temperature Spin down RNase A vial briefly Add the RNase A solution to buffer Al and mix well before use e Add 8 mL PD1222 00 or 60 mL PD1222 01 or 96 mL PD1222 02 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 Buffer N3 may form precipitates below 10 C warm up at 37 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 Materi
9. rol 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 OD o9 Buffers need to be scaled up if over amount of cultures are being processed 2 Harvest the bacterial culture by centrifugation for 1 minute at 13 000 rpm Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium Remove the residue medium completely Note Residue medium will cause e Poorcell lysis and thus lower DNA yield e Loose pellet after centrifugation in step 6 3 Add 400 uL Buffer A1 and completely resuspend bacterial pellet by vortexing or pipetting Complete resuspension is critical for bacterial lysis and lysate neutralization 4 Add 450 uL Buffer B1 mix gently by inverting 10 times do not vortex and incubate at room temperature for 5 minutes Note Do not incubate for more than 5 minutes 5 Add 80 uL Buffer N3 mix completely by inverting shaking the vial for 5 times 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 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 7 Careful
10. uffer RET and 100 ethanol Additional buffers can be purchased from Biomiga 3 Use same volume of Wash Buffer DNA Wash Buffer and EndoFree Elution Buffer 4 Pre warm the Endofree Elution Buffer at 65 C and let the column stand for 5 min after adding Endofree Elution Buffer Biomiga EZgene EndoFree ezFlow Plasmid Miniprep Kit II Page 9 Trouble Shooting Guide running in agarose gel DNA doesn t freeze or smell of ethanol from column 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 2 M NaOH and 1 SDS Low Yield Bacterial culture Grow bacterial 12 16 hours Spin down overgrown or not fresh cultures and store the pellet at 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 according to plasmid instructions 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 after contamination after adding Buffer B1 adding Buffer B1 Do not incubate more than 5 minutes after adding Buffer B1 RNA contamination RNase A not added to Add RNase A to Buffer Al Buffer Al Plasmid DNA floats Ethanol traces not Make sure that no ethanol residual out
11. w 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 5 10 minutes Transfer the column to an endofree 1 5 mL tube and add 50 100 uL of EndoFree Elution Buffer Incubate for 1 minute and centrifuge at 13 000 rpm for 1 minute to elute DNA Reload the eluate into the column use the same 1 5 mL tube and incubate for 1 minute centrifuge at 13 000 rpm for 1 minute to elute DNA Note The eluted DNA is ready for transfection of endotoxin sensitive cell lines primary cultured cells or microinjection Page 8 Biomiga EZgene EndoFree ezFlow 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 10 mL for miniprep kit 2 Use2x volumes of the Buffer A1 Buffer B1 Buffer N3 Buffer RET and 100 ethanol Additional buffers can be purchased from Biomiga 3 Usesame volume of DNA Wash Buffer and Endofree Elution Buffer Purification of plasmid gt 12 kb For isolating plasmid DNA 12 kb use the following guideline Culture volume Use 2 x volumes of the culture 2 Use2 x volumes of the Buffer A1 Buffer B1 Buffer N3 B
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