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1. 13 Centrifuge the column with the lid open at 13 000 rpm for 2 minute Note It is critical to removes ethanol residues completely The remaining ethanol will inhibit the elution of DNA from the column 14 Transfer the column to an endofree 1 5 mL tube and add 50 100 uL of ddH O or Endofree Elution Buffer Supplied 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 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 ddH O Note The DNA is ready for transfection of endotoxin sensitive cell lines primary cultured cells or microinjection 15 Determination of DNA concentration Concentration ug mL OD nm x 50 x dilution factor Note Two elutions give rise to maximum DNA yield Use less EndoFree Elution Buffer if high concentration is desired Page 8 Biomiga EZgene EndoFree Plasmid Miniprep Kit A2 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 9 in previous protocol on page 6 and 7 Carefully transfer the clear lysate from step 9 in the previous protocol to a DNA column and turn on the vacuum to allow the lysate pass through the column Optional Add 500 uL Buf
2. Or add 200 uL Chloroform 37 C vortex to mix well repeat step 8 Note Up to 99 of the endotoxin can be removed by extracting with the EndoClean Buffer once Another extraction is necessary if less than 0 1 EU Endotoxin ug of DNA is desired by repeating step 7 8 Transfer the clear phase lysate avoid the colored phase to a clean 2 0 mL Add 450 uL of Buffer N3 and 400 uL of 100 ethonal Mix well by sharp hand shaking for 3 times Transfer 700 uL of the lysate ethanol mixture to a DNA column and centrifuge at 13 000 rpm for 20s Decant the flow through liquid and insert the column back to the collection tube Transfer the remaining solution to the column and centrifuge at 13 000 rpm for 20s Decant the flow through liquid and insert 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 Biomiga EZgene EndoFree Plasmid Miniprep Kit Page 7 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 for details 12 Add 650 uL of DNA Wash Buffer and centrifuge at 13 000 rpm for 20s Decant the flow through liguid and insert the column back to the collection tube Repeat step 12
3. EndoFree 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 3 Culture volume Use 2 x volumes of the high copy number culture Use up to 25 mL for miniprep II Use 2 x volumes of the Buffer A1 Buffer B1 Buffer N3 and Buffer KB Additional buffers can be purchased from Biomiga Use same volume of DNA Wash Buffer and Endofree 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 Use 2 x volumes of the Buffer A1 Buffer B1 Buffer N3 and Buffer KB Additional buffers can be purchased from Biomiga Use same volume of DNA Wash Buffer and Endofree Elution Buffer Pre warm the Endofree Elution Buffer at 65 70 C and let the column stand for 5 mins after adding Endofree Elution Buffer Biomiga EZgene EndoFree 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 adding Buffer B1 e Make fresh Buffer B1 if the cap had not been closed tightly Buffer B1 0 2N NaOH and 1 SDS Low Yield Bacterial culture Grow bacterial 12 16
4. mix the solution well if the mixture still appears conglobated brownish or viscous more mixing is reguired to completely neutralize the solution Page 6 Biomiga EZgene EndoFree Plasmid Miniprep Kit 10 11 Centrifuge the lysate at 12 000 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 Carefully transfer the clear lysate into 2 0 mL tube and add 0 1 volume of EndoClean Buffer Mix by vortexing till homogeneous and incubate on ice for 10 minutes Mix by tapping the tube several times during incubation Note The solution becomes red and turbid after adding EndoClean Buffer The solution becomes clear after incubation on ice Note Use a serological pipet or a tip cut with a clean razor in the end to transfer the EndoClean Buffer Note Mix the sample several times during incubation without leaving ice Centrifuge the solution at 12 000 rpm for 10 minutes at room temperature the temperature must be greater than 23 C The solution should separate into 2 phases the upper clear phase contains DNA and the lower organic phase contains endotoxin The two phases will not separate if the temperature is less than 23 C Note If phase partitioning is not observed after centrifugation Incubate the solution at 65 C for 5 minutes The solution becomes turbid again And then repeat step 8
5. 15 on page 9 10 Note The plasmid DNA is purified The following steps are for removal of endotoxin After the plasmid is purified add 0 1 volume of EndoClean Buffer to the plasmid sample in a 2 mL centrifuge tube For example add 10 pL EndoClean Buffer to 100 uL plasmid sample The solution becomes turbid after adding EndoClean Buffer If the temperature is below 20 C the solution remains clear Vortex the sample for 10s and incubate the tube on ice for 10 min Mix the sample several times without leaving ice The solution becomes clear after incubating on ice Centrifuge at 12 000 rpm at room temperature for 10 minutes the temperature must be greater than 23 C for phase partitioning Carefully transfer the upper clear layer solution to another 2 mL tube Note If phase partitioning is not observed after centrifugation add 200 pL Chloroform votex for 10s and repeat step5 Precipitate plasmid DNA with 0 1 volume of 3 M KAc pH 5 2 and 0 7 volume of Isopropanol Centrifuge at 12 000 rpm for 10 min Carefully decant the supernatant Add 1 mL 70 ethanol and centrifuge at 12 000 rpm for 5 min Carefully decant and air dry the DNA for 30 minutes in a hood Resuspend the DNA with ddH5O or Endofree Elution Buffer Supplied Note The DNA is ready for downstream applications such as cloning subcloning RFLP Library screening in vitro translation seguencing transfection and microinjection Page 10 Biomiga EZgene
6. 4 16 hours with vigorous shaking 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 gpog Buffers needs to be scaled up if over amount of cultures are being processed Note Do not use a starter culture that has been stored at 4 C Note Do not grow starter culture directly from glycerol stock Note The culture can be centrifuged 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 2 Harvest bacterial culture by centrifugation for 1 minute at 10 000 x g Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium Remove the residue medium completely 3 Add 450 uL Buffer Al and completely resuspend bacterial pellet by vortexing or pipetting Complete resuspension is critical for optimal yields 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 Note Buffer B1 precipitates cloudy look below room temperature Warm up Buffer BI at 50 C to dissolve precipitation before use 5 Add 100 uL Buffer N3 mix completely by inverting shaking the vial for 5 times and sharp hand shaking for 2 times Note It is critical to
7. Contents IntroductiOmn oio bes oret YANA ND Abest aa nanah 2 Important Notes ih 2 Storage and Stability sese n 4 Before Starting ose oa NAIA an Nan 5 Kit COntentS an REED sn 5 Safety Information naa 5 EZgene EndoFree Plasmid Miniprep Spin Protocol 6 A Removal of Endotoxin during Plasmid Purification 6 B Removal of Endotoxin after Plasmid Purification 9 EZgene EndoFree Plasmid Miniprep Spin Vacuum Protocol 10 Purification of Low Copy Number Plasmid and Cosmid 11 Trouble Shooting Guide ooooo oom WWW 12 Related Product ir O e ntact gd dw HY an ana 13 Biomiga EZgene EndoFree Plasmid Miniprep Kit Page 1 Introduction Key to this kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our matrix while proteins and other impurities are removed by wash buffer Nucleic acids are easily 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 emdotoxin level to 0 1 EU Endotoxin per
8. fer KB into the spin column and allow the buffer pass the column by vacuum 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 Reference Table 2 on page 3 Add 650 uL of DNA Wash Buffer to the column and allow the vacuum to draw the liguid 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 Transfer the column to an endofree 1 5 mL tube and add 50 100 uL of ddH O or Endofree Elution Buffer Supplied 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 DNA is ready for transfection of endotoxin sensitive cell lines primary cultured cells or microinjection Biomiga EZgene EndoFree Plasmid Miniprep Kit Page 9 EZgene EndoFree Plasmid Miniprep Protocol B Removal of Endotoxin affer Plasmid Purification This protocol is designed for removing the endotoxin after the plasmid is purified 1 2 Follow the protocol on Page 6 from Step 1 to 6 Carefully transfer the clear lysate to a 1 5 mL tube and add 500 uL Buffer N3 and 400 uL 100 ethanol Mix well by sharp hand shaking for 2 times and go to step 10
9. 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 according plasmid to instructions on page 14 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 BI incubate more than 5 minutes after adding Buffer B1 RNA contamination RNase A not added Add RNase A to Buffer Al to Buffer 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 elute the plasmid DNA Re gel DNA doesn t centrifuge or vacuum again if freeze or smell of necessary ethanol Page 12 Biomiga EZgene EndoFree Plasmid Miniprep Kit Related Products Catalog Product Name Preps Price 5 PD1211 02 250 220 00 PD1213 02 250 250 00 PD1811 02 96 well plasmid mini kit 20x96 2000 00 Biomiga EZgene EndoFree Plasmid Miniprep Kit Page 13 ga EZg
10. nd 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 Table2 endA strains of E Coli Stra DH5a DH21 DHI JMI06 JM109 SK2267 SRB XLO TOPIO DHIOB JM103 IM107 SK1590 MM294 stbiz EL BIS182 DH20 JMI05 JMIOS SK1592 Select96 stpm ANID C600 JM110 RRI ABLE C CJ236 KW251 P2392 BL21 DE3 HB101 TGI TBI ABLE K oa LE392 PR700 ca JM101 JM83 TKBI HMS174 ES1301 M1061 Q358 BMH 71 18 All NM strains All Y strains Optimal Cell Mass OD 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 55 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 ODgpgg 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 ODaoo is 3 0 the optimal culture volume should be 10 15 mL Culture Volume Use a flask or tube 4 times bigger in volumn than the culture medium to secure optimal condition for bacteria gr
11. owth 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 EndoFree Plasmid Miniprep Kit Page 3 Before Starting Alternative endotoxin removal procedures are provided Protocol A removes endotoxin during the purification of plasmid DNA while 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 RNase A It is stable for half of year under room temperature Spin down RNase A vial briefly Add the RNase A solution to Buffer Al and mix well before use Add 8 mL PD1212 00 or 60 mL PD1212 01 or 96 mL PD1212 02 96 100 ethanol to each DNA Wash Buffer bottle before use Buffer B1 precipitates below room temperature It is critical to warm up the buffer at 50 C to dissolve the precipitates before use Keep the cap tightly closed for Buffer B1 after use Ensure the availability of centrifuge capable of 13 000 rpm Carry out all centrifugations at room temperature Materials supplied by use
12. rs e 96 100 ethanol e 1 5 mL and 2 0 mL pyrogen free microcentrifuge tubes e High speed microcentrifuge e Vacuum manifold if vacuum protocol is applied Page 4 Biomiga EZgene EndoFree Plasmid Miniprep Kit Kit Contents Catalog PD1212 00 PD1212 01 PD1212 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 4mL 30 mL 175 mL Buffer KB 3 mL 30 mL 135 mL DNA Wash Buffer 2mL 15mL 3 x 24 mL EndoClean Buffer 1 mL 10 mL 40 mL Endofree Eluiton Buffer 1 mL 10 mL 30 mL RNase A 0 25 mg 2 5 mg 12 5 mg 20 mg mL 12 5 uL 125uL 625 uL User Manual 1 1 1 Add 8 mL PD1212 00 or 60 mL PD1212 01 or 96 mL PD1212 02 96 100 ethanol to each DNA Wash Buffer bottle before use Safety Information e Buffer N3 contains acidic acid wear gloves and protective eyewear when handling e Buffer N3 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 EndoFree Plasmid Miniprep Kit Page 5 EZgene EndoFree Plasmid Miniprep Protocol A Removal of Endotoxin during Plasmid Purification This protocol is designed for removing the endotoxin during the plasmid purification A1 Spin Protocol 1 Inoculate 3 12 mL LB containing appropriate antibiotic with a fresh colony Grow at 37 C for 1
13. ug of plasmid DNA The endofree 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 miniprep column has a plasmid 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 contact our customer service for further information and reference the Table 1 for the commonly used plasmids Table 1 Commonly used plasmids 1 Expected Yield Plasmid Origin Copy Numbers woe 10 mL pSC101 pSC101 5 0 5 0 75 pACYC P15A 10 12 1 1 5 pSuperCos pMB1 10 20 1 2 5 pBR322 pMB1 15 20 1 5 2 5 pGEMR Muted pMB 1 300 400 30 40 pBluescript ColE1 300 500 30 50 pUC Muted pMB 1 500 700 50 70 Page 2 Biomiga EZgene EndoFree Plasmid Miniprep Kit Host Strains The strains used for propagating plasmid have significant influence on yield Host strains such as Top 10 a

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