Kit Manual
Contents
1. ABLE pai2s LE392 pr700 BL2I DES K pLysS 1101 Ms3 TkBi V5175 gsisor Mio61 Q358 IH 7I All NM strains All Y strains Optimal Cell Mass OD o 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 ODgo9 A high ratio of biomass over lysis buffers result in low DNA yield and purity Culture Volume Use a flask or tube with a volume at 4 times 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 Table 3 The optimal cell mass culture Volume and Binding Capacity for the mega DNA units DNA Units Mega 3 Mega 6 Mega 10 Optimal Cell Mass 1200 2500 4500 Culture Volume 500 mL 1000 mL 1500 mL Binding Capacity 3 4 mg 6 7 mg 10 12 mg 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 Plasmid ezFlow ezFilter Mega 3 Kit Page 3 Before Starting Prepare all components and get all necessary materia2. closed tightly Buffer B1 0 2N 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 to 2 x of plasmid original volume Increase the volume of buffer Al Bl N3 according to instruction on page 8 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 solution 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 running in agarose gel DNA doesn t freeze or smell of ethanol completely removed from column remaining in the silicon membrane before eluting the plasmid DNA Re vacuum again if necessary Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit Page 11
3. 5 mL 130 mL 2x330 mL RNase A 20 mg mL uL 5 uL dosi Endofree Elution Buffer 20 mL 40 mL 200 mL User Manual Safety Information e Buffer N3 contains acetic acid use gloves and protective eyewear when handling e Buffer N3 Buffer 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 Plasmid ezFlow ezFilter Mega 3 Kit Page 5 EZgene Plasmid ezFilter Megaprep 3 Protocol 1 Inoculate 500 mL LB containing appropriate antibiotic with 500 uL fresh starter culture Grow at 37 C for 14 16 h 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 h with vigorous shaking 250 rpm The buffer volumes need to be scaled up if processing over 500 mL of culture Harvest 500 mL overnight bacterial cells by centrifugation at 5 000 x g for 10 minutes at room temperature Decant or aspirate medium and discard Note Remove the residual medium completely for optimal cell lysis and neutralization Resuspend the bacterial pellet in 30 mL Buffer A1 Add RNase A to Buffer A1 before use Pipet or vortex till the bacterial pellet dispersed thoroughly Complete resuspension is critical for optimal yields Then add 1 5 mL B
4. Contents odi EET 1 Intt duction cene ded bed plu eme heed ver EH PEE Ee nen 2 Important Notes ec ced eim e DE ee ege mee eere tees 2 Storage and Stability cece cee cece eee e eee ee eee e eens mene 3 Before Starting Lease cce eee E Entre epe deme Ret eaa ete duane e 4 Kit COMTEMES 5x ose eter A Ee eae EE P Rae REESE 5 Safety Information io cios ecce ner tee e e e a etes 5 EZgene Plasmid ezFilter Megaprep 3 Protocol 6 Purification of Low Copy Number Plasmid Purification 9 Trouble Shooting Guide sss 10 Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 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 contaminates are removed under certain optimal conditions Nucleic acids are easily eluted with sterile water or elution buffer Unlike other procedures our patented plasmid purification kit has no guanidine salt in the buffer the purified DNA is guanidine ion exchange resin residues free which enable the high performance of downstream applications such as transfection restriction mapping library screening sequencing as well as gene therapy and genetic vaccinations 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
5. copy number plasmid purification For low copy number plasmids both the culture volume and the buffer volume need to be scaled up 2 times Reference Table 1 for the commonly used plasmids Table 1 Commonly used plasmids Plasmid Origin Copy Numbers Expected Yield ug per 500 mL pSC101 pSC101 5 50 60 pACYC PISA 10 12 80 100 pSuperCos pMBI 10 20 80 150 pBR322 pMB1 15 20 100 150 pGEM Muted pMB 1 300 400 2000 2500 pBluescript ColE1 300 500 2000 3000 pUC Muted pMB 1 500 700 3000 4000 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 For purifying plasmid DNA from endA strains Table 2 we recommend use product PD1714 Page 2 Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit Table2 endA strains of E Coli DHSa_ DHI DH2 JM106 JM109 SK2267 SRB XLO TOPIO DHIOB JM103 IM107 SK1590 MM294 stbi2 Xt BI5182 DH20 IMIOS IMIOS SK1592 Select96 stblaw S ABLE co00 JMII0 RRI C C3236 KW251 P2392 BL21 DE3 HB101 rG1 Tgi
6. hen all the lysate pass through the DNA unit vacuum for minute Wash the DNA membrane with 50 mL 70 ethanol and vacuum for 1 minute at maximum force Wash the DNA membrane with another 50 mL 70 ethanol and vacuum for 1 minute at maximum force Add 50 mL 10046 ethanol evenly to the DNA membrane and vacuum for 1 minute Turn off the vacuum wait for 1 minute and discard the liquid waste in the bottle Reconnect the bottle to the DNA binding unit Turn on the vacuum Page 8 Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit 13 14 15 for 20 minutes at maximum force It is critical to dry the residual ethanol for optimal yield Turn off the vacuum wait for 1 minute and replace the 500 mL or 1 000 mL standard bottle with a sterile 50 mL conical tube screw tight Add 10 mL Endofree Elution Buffer evenly to the membrane and incubate for 2 minutes Turn on vacuum to elute DNA Typically 3 5 mL of solution can be collected This is the 1 elution Turn off the vacuum and replace the 50 mL conical tube with another sterile 50 mL conical tube screw tight Add 5 mL Endofree Elution Buffer and incubate for 1 minute Turn on the vacuum and collect the 2 elution typically 2 4 mL of solution can be collected Note The DNA is ready for downstream applications such as transfection of endotoxin sensitive cell lines primary cultured cells or microinjection Note Two elutions give rise to maximum DNA yield For maxi
7. ls ready by examining this instruction booklet and become familiar with each steps Important v 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 A1 and mix well before use Buffer ER should be stored at 4 C Buffer B1 precipitates below room temperature It is critical to warm up the buffer at 50 C to dissolve the precipitates before use Buffer N3 may form precipitates below 10 C warm up at 37 C to dissolve the precipitates before use Keep the cap tightly closed for Buffer B1 after use Make sure the availability of centrifuge and vacuum manifold especially after mixing the lysate with ethanol the sample needs to be processed immediately by vacuum Materials supplied by users v v v v 70 ethanol and 100 ethanol Pump driven vacuum system 1 000 mL bottle Corning 430518 or 430282 or equivalent pyrex glass bottles 50 mL conical tubes High speed centrifuge tube for endotoxin removal if desired Page4 M Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit Kit Contents Catalog PD1621 00 PD1621 01 PD1621 02 Preps 1 2 10 DNA Unit 1 2 10 Filter Unit 1 2 10 Replacement Cup 1 2 10 Buffer Al 35 mL 70 mL 350 mL Buffer ER 1 8 mL 3 5 mL 17 5 mL Buffer B1 35 mL 70 mL 350 mL Buffer D1 3 5 mL 7 mL 35 mL Buffer N3 10 mL 20 mL 100 mL Buffer RET 6
8. mum yield and higher concentration pool the elutions together add 0 1 volume 3M Potassium Acetate or Sodium acetate pH 5 2 and 0 7 volume isopropanol Centrifuge at top speed for 10 min Discard supernatant Wash the DNA with 1000 uL 70 ethanol centrifuge for 5 min carefully decant Air dry the pellet for 10 20 minutes in a tissue culture hood Resuspend the DNA in Endofree Elution Buffer Note Use less Endofree Elution Buffer if high concentration is desired DNA concentration ug mL OD26 nm x 50 x dilution factor Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit Page 9 Purification of Low Copy Number Plasmid and 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 volume of the high copy number culture 2 Use 2 x volume of the Buffer Al Buffer B1 and Buffer N3 and 100 ethanol Additional buffers can be purchased from Biomiga 3 Use same volume of wash buffer 70 ethanol and 100 ethanol and Endofree Elution Buffer Page 10 Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit Trouble Shooting Guide Problems Possible Reasons Suggested Improvements Low Yield Poor Cell lysis e Resuspend pellet thoroughly by votexing and pipeting prior adding Buffer B1 e Make fresh buffer B1 if the cap had not been
9. the filter unit This will speed up the flow rate of the filter unit Normally around 200 mL lysate can be filtered through the filter unit within 20 30 minutes Pour the remaining white precipitates to the filter unit when most of the lysate has been filtered through Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit Page 7 10 11 12 Figure 1 Instruction of filter assembling Filter Assembling Right Wrong wv x Wi Ring Note 3 If the flow through gets too slow turn off the vacuum and wait for 1 minute Carefully detach the upper filter cup and replace it with the replacement cup Assemble the unit as Figure 1 Pour the lysate from the original cup to the replacement cup Turn on the vacuum and filter the rest of the lysate When most of the lysate has been filtered through the unit turn off the vacuum wait for 1 minute detach the unit and discard the upper filter cup including the rubber rings Note The DNA is in the collection bottle Connect the DNA unit to a 500 mL or 1 000 mL standard bottle and screw tight Connect the DNA unit to the vacuum with the vacuum off Add 1 volume of Buffer RET For example 60 mL of Buffer RET to 60 mL of clear lysate and add 36 mL 100 ethanol to the lysate bottle Mix well by sharp hand shaking 3 5 times and immediately pour half of the lysate ethanol mixture to the DNA unit and turn on the vacuum Pour the rest of the lysate ethanol mixture into the DNA unit W
10. uffer ER into the suspended bacterial culture Mix well by inverting 5 10 times Add 27 mL Buffer B1 Mix gently but thoroughly by inverting 10 times and incubate at room temperature for 5 minutes to obtain a cleared lysate Do not incubate longer than 5 minutes Over incubating causes genomic DNA contamination and plasmid damage Avoid vigorous mixing as this will shear the genomic DNA Then add 3 mL Buffer D1 mix gently and incubating for another 5 minutes Add 8 mL Buffer N3 and mix immediately by inverting 5 times till a flocculent white precipitate forms Vortex the lysate for 5 seconds Note It is critical to mix the lysate well if the mixture still appears conglobated brownish or viscous more mix is required to completely neutralize the solution Attach the 2 layer filter unit to a sterile 500 mL or 1000 mL standard bottle Corning 430518 or 430282 or equivalent pyrex glass bottle and screw tight Connect the unit to a pump driven vacuum system Transfer the clear lysate from the bottom of the mixture use a 50 mL serological pipet to the filter unit Stand by for 5 minutes and turn on the vacuum with low vacuum force and increase to maximum vacuum force after 5 minutes Note 1 Low vacuum force prevents clogging of the filter membranes Note 2 Use a 50 mL serological pipet to transfer the relatively clear lysate from the Page 6 Biomiga EZgene EndoFree Plasmid ezFlow ezFilter Mega 3 Kit bottom of the lysate bottle to
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