Kit Manual
Contents
1. EndoFree Plasmid Midiprep Spin Protocol A Removal of Endotoxin during Plasmid Purification This protocol is designed for removing the endotoxin during the plasmid purification 1 Inoculate 15 50 mL LB containing appropriate antibiotic with 50 uL fresh starter culture Incubate at 37 C for 14 16 hours 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 hours with vigorous shaking 250 rpm 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 Do not use more than 50 mL culture or cell mass greater than 150 2 Harvest the bacterial by centrifugation at 5 000 x g for 10 minutes at room temperature Pour off the supernatant and blot the inverted tube on paper towels to remove residual medium 3 Add 2 5 mL Buffer Al Add RNase A to Buffer Al before use and completely resuspend bacterial pellet by vortexing or pipetting Complete resuspension is critical for optimal yields 4 Add 2 5 mL Buffer B1 mix gently but thoroughly by inverting 5 times and incubate for 5 minutes to obtain a slightly clear lysate Note Do not incubate longer than 5 minutes Over incubating causes genomic DNA contamination and plasmid damage 5 Add 600 uL Buffer N3 mix immediately by inverting 5 times and sha2. 200 pUC Muted pMB1 500 700 150 250 Page 2 Biomiga EZgene Endofree Plasmid Midiprep Kit Host Strains The strains used for propagating plasmid have significant influence on yield Host strains such as Top 10 and DHS 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 _For purifying plasmid DNA from endA strains Table 2 we recommend use product PD1712 Table 2 endA strains of E Coli EndA Strains of E Coli DHSa DHI DH21 JM106 JM109 SK2267 SRB XLO TOP10 DH10B JM103 JM107 SK1590 MM294 Stbl2 s XL10 BJ5182 DH20 JM105 JM108 SK1592 Select96 sto4 cig EndA Strains of E Coli C600 JM110 RRI ABLE C CJ236 KW251 P2392 BL21 DE3 HB101 TGl TBI ABLE K Pe LE392 PR700 BL21 DE3 M pLysS JM101 JM83 TKBI 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 mediums such as TB or 2xYT are used make sure the cell density doesn t exceed 3 0 OD o9 A h
3. Contents MntrodUCti Ons 4520 iocg anae a e a pede Tse beg a E AE 2 Important Notes iona eaa E E end Slwidia ecb ie sees 2 Storage and Stability 0 c cece cece eee e eee naeenae enna eees 3 Before Staring sueo i e cise sailed E EE E a sateen otieed 4 Kit Contents ose kersen pnt na n e a Ae E EEE 5 Safety Information cc cece cece ence eee eee eee eneeeeaeeenaeenees 5 EZgene EndoFree Plasmid Midiprep Protocol 465 6 A Removal of Endotoxin during Plasmid Purification 6 B Removal of Endotoxin after Plasmid Purification 8 Purification of Low Copy Number Plasmid and Cosmid 9 Trouble Shooting Guide eee eeesscsseceseceeeeeeeeneeeseeseeeseeeee ena eaee 10 Biomiga EZgene Endofree Plasmid Midiprep Kit 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 impurities are removed by wash buffer Nucleic acids are easily eluted with sterile water or Elution Buffer Unlike other kits in the markets our patented plasmid purification kit has no chaotropic salts in the buffer The purified DNA is guanidine anion exchange resin residues free Plasmid isolated with traditional protocol normally contains high level of endotoxins Lipopolysaccharides or LPS For transfection of endotoxin sensitive cell lines or microinjection the endot
4. Elution Buffer Note The DNA is ready for downstream applications such as cloning subcloning RFLP Library screening in vitro translation sequencing transfection and microinjection DNA concentration ug mL OD260 nm X 50 x dilution factor 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 up to 100 mL for midipreps Use 2 x volumes of the Buffer A1 Buffer B1 Buffer N3 and 100 ethanol Additional buffers can be purchased from Biomiga Use same volumes of Wash Buffer 70 ethanol and Endofree Elution Buffer Biomiga EZgene Endofree Plasmid Midiprep Kit Page 9 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 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 and increase the plasmid vol
5. igh ratio of biomass over lysis buffers result in low DNA yield and purity The midi column has an optimal biomass of 100 150 For example if the OD oo is 3 0 the optimal culture volume should be 25 50 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 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 Midiprep Kit Page 3 Before Starting Alternative 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 step Important Notes RNase A It is stable for half a year under 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 bef
6. ning is not observed after centrifugation Incubate the solution at 65 C for 5 minutes The solution becomes turbid again And then repeat step 8 Or add 200 uL Chloroform 37 C 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 Carefully transfer the clear supernatant into a 15 mL conical tube avoid the interface precipitates Add 3 mL Buffer N3 and 3 mL 100 ethanol Mix immediately by sharp hand shaking for 5 times The mixture of ethanol lysate needs to be transfer to the DNA column immediately Immediately transfer 6 mL the lysate ethonal mix into a DNA column with a 15 mL collection tube Centrifuge at gt 2 500 x g for 1 min at room temperature Remove the column from the tube and discard the flow through liquid Reinsert the column to the collection tube Repeat step 10 till all the lysate ethonal mix has been passed through the column Optional Add 3 0 mL Buffer KB into the spin column centrifuge at gt 2 500 x g 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
7. ore use Keep the cap tightly closed for Buffer B1 after use Make sure the availability of centrifuge especially after mixing the lysate with ethanol the sample needs to be processed immediately by centrifugation Centrifuge speed at 5 000 x g is recommended In case the collection tube doesn t fit in high speed centrifuge rotor use benchtop centrifuge and spin at 2 500 x g with double centrifugation time Carry out all centrifugations at room temperature Materials supplied by users 70 ethanol and 100 ethanol High speed centrifuge and 30 mL high speed centrifuge tubes 15 mL and 50 mL conical tubes Page 4 Biomiga EZgene Endofree Plasmid Midiprep Kit Kit Contents Catalog PD1415 00 PD1415 01 PD1415 02 Preps 2 10 25 EzBind Columns 2 10 25 Buffer Al 6 mL 30 mL 70 mL Buffer B1 6 mL 30 mL 70 mL Buffer N3 8 mL 40 mL 100 mL Buffer KB 7 mL 35 mL 85 mL EndoClean Buffer 2 mL 10 mL 25 mL RNase A 0 6 mg 3 mg 7 mg 20 mg mL 30 uL 150 uL 350 uL Endofree Eluiton Buffer 3 mL 15 mL 50 mL User Manual 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 Midiprep Kit Page 5 EZgene
8. otocol is designed for removing the endotoxin after the plasmid is purified 1 2 Follow the protocol from Step 1 to 6 on page 6 7 Transfer the lysate to a clean 15 mL conical tube and add 3 mL of Buffer N3 and 3 mL of 100 ethanol mix well and go to step 10 15 on page 9 10 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 0 1 mL EndoClean Buffer to 1 mL plasmid sample The solution becomes turbid after adding EndoClean Buffer Vortex the tube for 5s and put on ice for about 10 minutes Mix the sample several times without leaving ice The solution becomes clean after incubating on ice Page 8 Biomiga EZgene Endofree Plasmid Midiprep Kit Centrifuge at 12 000 x g at room temperature for 10 minutes the temperature must be greater than 23 C for phase partitioning Note If phase partitioning is not observed after centrifugation Incubate the solution at 65 C for 5 minutes and repeat step 5 Or add 200 uL Chloroform 37 C votex for 10s and repeat step 5 Carefully transfer the upper clear layer solution to a 2 mL tube Precipitate plasmid DNA with 0 1 volume of 3 M KAc pH 5 2 and 0 7 volume of Isopropanol Centrifuge at 12 000 x g for 10 minutes Carefully decant Add 1 mL 70 ethanol and centrifuge at 12 000 x g for 5 minutes Carefully decant and air dry the DNA for 30 minutes in a hood Resuspend the DNA with Endofree
9. oxins 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 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 15 to 50 mL of E coli culture The midi column has a DNA binding capacity of 250 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 Table 1 for the commonly used plasmids Table 1 Commonly used plasmid ees Expected Yield Plasmid Origin Copy Numbers T o 50 mL pSC101 pSC101 5 5 pACYC PISA 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
10. page 3 Add 5 mL 70 ethanol into the column centrifuge at gt 2 500 x g for 1 min Biomiga EZgene Endofree Plasmid Midiprep Kit Page 7 14 15 16 Remove the column from the tube and discard the flow through Reinsert the column into the collection tube Repeat step 13 Centrifuge the column with the lid open at gt 2 500 x g for 10 minutes This step removes residual ethanol for optimal elution in next step Note Residual ethanol can be removed more efficiently with the column lid open High centrifuge speed is suggested to remove the ethanol It is critical to remove residual ethanol completely Carefully transfer the spin column into a clean 15 mL tube and add 0 5 mL Endofree Elution Buffer to the center of the column and incubate for 1 minute at room temperature Elute the DNA by centrifugation at gt 2 500 x g for 5 minutes If higher yield is desired reload the eluate in the 15 mL tube to the column incubate for 1 minute and centrifuge again Note Two elutions give rise to maximum DNA yield Use less Endofree Elution Buffer if high concentration is desired Note The DNA is ready for downstream applications such as cloning subcloning RFLP Library screening in vitro translation sequencing transfection and microinjection The DNA concentration can be calculated as follows DNA concentration ug mL OD 601m X 50 x dilution factor B Removal of Endotoxin after Plasmid Purification This pr
11. rp hand shaking for 5 times Note 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 6 Transfer the lysate to a high speed centrifuge tube and centrifuge at 13 000 x g for 10 minutes at room temperature Note Syringe filter Supplied in PD1416 or purchase separately from Biomiga could be used to filtrate the lysate if high speed centrifuge is not available 7 Transfer the clear lysate to a new high speed centrifuge tube and add 0 1 volume of EndoClean Buffer vortex for 10s and incubate on ice for 10 Page 6 Biomiga EZgene Endofree Plasmid Midiprep Kit 10 11 12 13 minutes Mix the sample several times without leaving ice Note Use a serological pipet or a tip cut with a clean razor in the end to transfer the EndoClean Buffer Note At room temperature gt 23 C the sample becomes turbid after adding EndoClean Buffer The solution becomes clear after incubating on ice Centrifuge at 13 000 x g for 10 min Alternatively the sample can be processed in a 15 mL conical tube and centrifuge at 2 500 x g for 15 min 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 partitio
12. ume of Buffer Al B1 N3 and 100 ethanol according to 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 A1 Buffer Al 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 gel from column before eluting the plasmid DNA Re DNA doesn t freeze or centrifuge or vacuum again if necessary smell of ethanol FOR RESEARCH USE ONLY Page 10 Biomiga EZgene Endofree Plasmid Midiprep Kit
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